The division's first major operation was to help relieve the Siege of Plei Me near Pleiku and the pursuit of the withdrawing People's Army of Vietnam (PAVN) which culminated in the Battle of Ia Drang, described in the book We Were Soldiers Once...And Young, was also the basis of the film We Were Soldiers. Because of that battle the division earned the Presidential Unit Citation (US), the first unit to receive such in the war. In 1966, the division attempted to root the communist Viet Cong (VC) and PAVN out of Bình Định Province with Operation Masher, Operation Crazy Horse and Operation Thayer. 1967 was then spent conducting Operation Pershing, a large scale search and destroy operation of PAVN/VC base areas in II Corps in which 5,400 PAVN/VC soldiers were killed and 2,000 captured. In Operation Jeb Stuart, January 1968, the division moved north to Camp Evans, north of Hue and on to Landing Zones Sharon and Betty, south of Quang Tri City, all in the I Corps Tactical Zone.

In the early morning hours of 31 January 1968, the largest battle of the Vietnam War, the Tet Offensive, was launched by 84,000 PAVN/VC soldiers across South Vietnam. In the Division's area of operation, the PAVN/VC forces seized most of the city of Huế. As the 3rd Brigade, 1st Cavalry Division, fought to cut off PAVN/VC reinforcements pouring into Huế, at Quang Tri City, five battalions, most from the 324th Division, attacked the city and LZ Betty (Headquarters 1st Brigade). To stop allied troops from intervening, three other PAVN/VC infantry battalions deployed as blocking forces, all supported by a 122mm-rocket battalion and two heavy-weapons companies armed with 82mm mortars and 75mm recoilless rifles. After intense fighting, 900 PAVN/VC soldiers were killed in and around Quang Tri City and LZ Betty. However, across South Vietnam, 1,000 Americans, 2,100 South Vietnamese, 14,000 civilians, and 32,000 PAVN/VC were killed.

In March 1968 the Division shifted forces to LZ Stud, the staging area for Operation Pegasus to break the siege of the Marine's Khe Sanh Combat Base—the second largest battle of the war. All three brigades participated in this airmobile operation, along with a Marine armor thrust. US Air ForceB-52s alone dropped more than 75,000 tons of bombs on PAVN soldiers from the 304th and 325th Divisions encroaching the combat base in trenches. As these two elite enemy divisions, with history at Dien Bien Phu and the Ia Drang Valley, depleted, the Division leapfrogged west, clearing Route 9, until at 0:800 hours 8 April, the 2nd Battalion, 7th Cavalry, linked-up with Marines at the combat base, ending the 77-day siege.

On 19 April 1968, as the 2nd Brigade continued pushing west to the Laotian border, the 1st and 3rd Brigades (about 11,000 men and 300 helicopters) swung southwest and air assaulted A Shau Valley, commencing Operation Delaware. The PAVN was a well-trained, equipped, and led force. They turned A Shau into a formidable sanctuary —complete with PT76 tanks; powerful crew-served 37mm antiaircraft cannons, some radar controlled; twin-barreled 23mm cannons; and scores of 12.7mm heavy machine guns. A long-range penetration operation was launched by members of the Division's long-range reconnaissance patrol (LRP) against the PAVN when they seized "Signal Hill"—the name attributed to the peak of Dong Re Lao Mountain, a densely forested 4,879 feet (1,487 m) mountain midway in the valley—so the 1st and 3rd Brigades, slugging it out hidden deep behind the mountains, could communicate with Camp Evans near the coast or with approaching aircraft.

Despite hundreds of B-52 and jet air strikes, the PAVN forces shot down a C-130, a CH-54, two Chinooks, and nearly two dozen UH-1 Hueys. Many more were lost in accidents or damaged by ground fire. The division also suffered more than 100 dead and 530 wounded in the operation. Bad weather aggravated the loss by causing delays in troop movements, allowing a substantial number of PAVN to escape to safety in Laos. Still, the PAVN lost more than 800 dead, a tank, 70 trucks, two bulldozers, 30 flamethrowers, thousands of rifles and machine guns, and dozens of antiaircraft cannons. They also lost tons of ammunition, explosives, medical supplies and foodstuffs.

In mid-May 1968 Operation Delaware ended, however, the division continued tactical operations in I Corps as well as local pacification and "medcap" (medical outreach programs to local Vietnamese). In the autumn of 1968, the Division relocated south to Phước Vĩnh Base Camp northeast of Saigon.^[12] In May 1970, the Division participated in the Cambodian Incursion, withdrawing from Cambodia on 29 June. Thereafter, the division took a defensive posture while US troops withdrawals continued from Vietnam. On 29 April 1971 the bulk of the division was withdrawn to Fort Hood, Texas, but its 3rd Brigade remained as one of the final two major US ground combat units in Vietnam, departing 29 June 1972. However, its 1st Battalion, 7th Cavalry, Task Force Garry Owen, remained another two months.

In the Vietnam War, the Division suffered more casualties than any other army division: 5,444 men killed in action and 26,592 wounded in action. However, the First Marine Division suffered 7,012 men killed in action and the Third Marine Division suffered 6,869 men killed in action.