The cathedral, like St. Patrick's in New York, plays an important symbolic role for Boston's Catholics, as it is the church associated with the archbishop and the archdiocese headquarters.
The statement from Massachusetts' four bishops was issued as both the state's highest court and the Legislature are considering the matter.
Word had spread through the Catholic community beginning last week that the archdiocese planned to have a statement read at Sunday's masses, so activists were ready when they heard the words they opposed read from the pulpit.
At the mention of the statement, about a dozen people, mostly men, stood up and faced the back of the church. Most of them then walked out, with three men walking out at the end of the homily.
Gay activists then issued their own statement that the church has "no moral authority" to advise on such matters in the wake of its sex abuse scandal.
"I feel angry," said Mark Murphy, 25, of Somerville, after walking out of the church. "I have to turn my back to the altar. I shouldn't have to do that."
During the homily at Holy Cross, Monsignor William H. Roche asked parishioners to read the marriage statement in the archdiocesan newspaper, The Pilot, and explained why the church opposes same-sex marriage.
The Massachusetts Supreme Judicial Court is considering whether the state Constitution allows same-sex marriage, and the Massachusetts Legislature is expected to consider a constitutional amendment that would define marriage as being solely between a man and a woman.
In his homily, Roche asked parishioners to talk to their lawmakers and support the traditional definition of marriage.
Roche said that it is impossible for same sex couples to fully experience marriage in the Catholic sense, but also said that Catholics should not be hostile to homosexuals. He later said he wasn't offended by the protest.
C.J. Doyle, executive director of the Catholic Action League of Massachusetts, called the protest "a bigoted expression of contempt for the Catholic bishops and the Catholic faith."
A Boston Archdiocese spokesman did not immediately return a phone call asking for comment.