Dota 2 was already the record holder, but the game broke another Steam record this weekend as 329,977 players were online simultaneously. The main reason perhaps that it was the final of the West Qualifier for The International 3 (basically the World Cup of Dota 2). Some of these players were likely just watching the action, but still, it's an impressive feat.
Dota 2 still remains in closed beta (even if beta keys are easy to come by) - with no word on when the beta may open up fully.