Cuba's Yuliesky Gurriel (left) and United States' Jason Donald
Defending champions Cuba defeated arch rivals the United States 5-4 via the new extra-innings rule at Wukesong Baseball Field.
The game was 3-3 after 10 innings, but Michel Enriquez hit a two-run double in the 11th inning to seal the win.
Cuba, seeking a fourth Olympic gold from five attempts, improved to 3-0 while the Americans slid to 1-2.
Debutants China stunned Taiwan 8-7, also via the extra-innings rule, to claim their first win of the Games.
Hou Fenglian dealt Taiwan a crushing blow by scoring three runs with a line drive to settle the game.
The new tie-break format was adopted this year for international competition in order to shorten games for television.
Teams put runners on first and second base and start from wherever they like in their batting order.
Defeat for the US leaves them struggling to make the semi-finals as only four teams will advance.
In night games, South Korea edged Canada 1-0 after pitcher Ryu Hyun-jin threw a complete-game shutout.
Japan beat the Netherlands 6-0, with Toshiya Sugiuchi pitching seven scoreless innings. Takahiro Arai hit runs in for Japan.