His first assignment was as a civilian chaplain at United States Military Academy which inspired him to enlist to become a military chaplain. In 1970, he officially became an Army Chaplain with the rank of Captain, serving with the 82nd Airborne Division. From 1971 to 1972, he served a tour of duty in the Vietnam War with the 173rd Airborne Brigade and 1st Cavalry Brigade. He then returned to Fort Gordon, Georgia for a year as Post Chaplain.
In 1973, he left the military for doctoral studies in Pontifical University of St. Thomas Aquinas (Angelicum), Rome. In 1976, he received a doctorate in sacred Theology for a thesis entitled The Origin and Development of Moral Principles in the Writings of Paul Ramsey. Subsequently, he returned to continue his service to the Archdiocese of New York. From 1983 to 1985 he served as the Secretary of Terence Cardinal Cooke and John Cardinal O’Connor.
In 1986, he was elevated to Monsignor. He served two terms as rector of St. Joseph's Seminary from 1985 to 1989 and 1994 to 1997. From 1990-1994, he served as Rector of the Pontifical North American College in Rome.
O'Brien was called "instrumental in Catholic seminary reform in the wake of clergy sex abuse disclosures", by The Baltimore Examiner.
He is regarded as having a genuine concern for the souls in his care. These traits were highlighted in the famous case of Eugene Hamilton, a seminarian struck with cancer in the course of his formation. As Hamilton’s one desire was to be ordained before he died, O’Brien’s intervention pushed the case to the Holy See, which green-lighted his petition for early priesthood. O'Brien ordained the 25 year-old in his family home hours before he died on January 25, 1997.
Some believed that O'Brien would be created a cardinal in the next consistory of November 2007. This did not occur. With the creation of Galveston-Houston as a new Cardinalitial See, it is questionable as to whether Baltimore will ever again receive a Cardinal archbishop. Only three of the fifteen men who have served as archbishop of Baltimore have been created Cardinals (James Gibbons, Lawrence Joseph Shehan, and William Henry Keeler) and none was elevated to Cardinal until he had served three or more years as Baltimore archbishop. During the press conference announcing his appointment at the Baltimore Basilica on July 12, 2007, Archbishop O'Brien said that: "“I am humbled in being named shepherd of this historic Premier See whose Catholic roots reach deep into our nation”. “Very much aware of the lengthy and impressive line of my predecessors, I pledge with God’s grace to give myself fully to the task of making Jesus Christ known by preaching his word and serving his people."
Commenting on O'Brien's appointment, The Baltimore Sun said in its front-page headline coverage on July 13, 2007, "He has leapt from military airplanes, served in jungles during the Vietnam War and travelled extensively to current battle zones in Afghanistan and Iraq. From his working-class roots...to the upper echelons of Catholic power – carrying a Christian message of peace and love to some of the world's worst war-torn terrain". The Baltimore Examiner described him as "Army jump school-qualified and troubleshooter for God at Catholic seminaries and remote Vietnam War firebases alike".
One of his "spiritual heroes", according to the Baltimore Sun, is St. Therese of Lisieux, the "Little Flower of Jesus".