In June 2010, O'Keefe was aboard the MV Mavi Marmara. During the Gaza flotilla raid, he was one of the passengers who disarmed IDF commandos boarding the ship. According to O'Keefe, he disarmed one from the 9mm pistol he was carrying, removing its bullets before hiding the pistol with the intention to keep it as evidence of the Israeli action against the ship. O'Keefe said he subsequently helped disarm another of the ship's IDF soldiers, by prising the commando's fingers from the rifle he was holding.
O'Keefe said of the experience that it was like "combat but without combat weapons". He said, "We had in our full possession, three completely disarmed and helpless commandos" who were "surrounded by at least 100 men ... [W]e could have done anything with them", adding that "woman provided basic first aid, and ultimately they were released, battered and bruised for sure, but alive. Able to live another day."
According to Greta Berlin, spokesperson for the Free Gaza Movement who was involved in the flotilla, O'Keefe "was responsible for some of the deaths on board the Mavi Marmara. Had he not disarmed an Israeli terrorist soldier, they would not have started to fire."
Ken O'Keefe was among those arrested and detained in Israel, where he (according to himself and another activist) was beaten at Tel Aviv airport when he resisted deportation while still in Israeli custody. He claims a policeman hit him on the head with a truncheon and that he was choked until he almost blacked out. He said he spent two more days in a detention facility in the airport after the incident. O'Keefe said the Irish consul general tried to convince him to agree to leave and asked him to wash the blood off his face but he refused.
A video showing his bloodied face was released upon his arrival in Istanbul. On June 6, 2010, the Israel Defense Forces (IDF) charged that O'Keefe is an "anti-Israel extremist" and "operative of the Hamas Terror organization". According to the IDF he was entering the Gaza Strip in order to "form and train a commando unit for the Palestinian terror organization." He responded: "If they had a supposed terrorist in their possession, why the hell did they let me go?" He acknowledged having had meetings with Prime Minister Ismail Haniyeh and other senior Hamas officials.