In January 1965, the 2nd Battalion, 1st Marines at Marine Corps Base Camp Pendleton, California deployed for a tour on Okinawa, Japan where they were redesignated the 3rd Battalion, 3rd Marines. At the time the Marines of 3rd Battalion expected a typical 13-month deployment followed by a quick return to the States. However, the battalion found itself caught up in the initial deployment of Marine units to Vietnam, and landed on May 12 along the coast south of Danang at an airfield called Chu Lai.

The battalion's first major operation was Operation Starlite where they teamed up with units of the 7th Marines. Operation Starlite was aimed at destroying the 1st Viet Cong (VC) Regiment, which was preparing to attack Chu Lai from the Van Tuong peninsula. The fighting began on August 18 when the battalion conducted an amphibious landing just east of the VC positions.

In October 1966, 3rd Battalion was deployed to combat the threat from the North Vietnamese Army in Quang Tri province. This would be a largely conventional fight that would continue into 1967. In December, the battalion suffered a tragic case of friendly fire when a pair of F-4 Phantoms dropped several bombs in the middle of Mike Company, killing seventeen Marines and wounding a dozen others. Corpsman Donald Rion was awarded a posthumous Silver Star for his efforts to treat the wounded, despite suffering a mortal wound himself.

In the spring of 1967, 3rd Battalion participated in a series of bloody engagements near Khe Sanh known as the Hill fights, culminating in the Battle of Hill 881, where 46 Marines from the battalion were killed. The violence in the area continued to increase throughout 1967, culminating in 1968's Tet Offensive. While deployed in Quang Tri, 3rd Battalion fought in such places as the Rockpile, Cam Lo, A-3, Gio Linh, Khe Sanh, and Con Thien.

In early 1969, 3rd Battalion 3rd Marines was sent south for several months to join Task Force Yankee in Operation Taylor Common near An Hoa. This three-month operation focused on destroying the primary base for North Vietnamese Army forces operating across several provinces and 3rd Battalion was awarded a Navy Unit Commendation for its actions during the operation. Ten Marines from 3rd Battalion were killed during the operation (out of 183 total US fatalities), and American forces captured numerous quantities of North Vietnamese armies and supplies. One Marine, Lance Corporal William R. Prom, was posthumously awarded the Medal of Honor for gallantry during Taylor Common.

In March, 1969, while M/3/3 was opcon to 1/4 at LZ Alpine, PFC Ronald L. Coker was posthumously awarded the Medal of Honor for gallantry in action. In July 1969, the battalion took part in Operations Virginia Ridge and Idaho Canyon, attempting to stop North Vietnamese infiltrators coming through the DMZ. The operation continued until September, when the 3rd Marine Regiment was ordered to stop operations in preparation for its redeployment back to the United States.

The battalion began to depart on October 7 and had fully arrived at MCB Camp Pendleton by the end of 1969. 3rd Battalion spent over 1,600 days in Vietnam and conducted 48 combat operations, the most of any Marine battalion in the conflict. 653 Marines who served in 3rd Battalion 3rd Marines lost their lives during the Vietnam War or were killed while operating with other units. Nearly 2,800 others were wounded.

During October and November 1969 the battalion relocated to MCB Camp Pendleton and was reassigned to the 5th Marine Amphibious Brigade.