An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein (CSAP). CSAP was single-chained, with an apparent molecular mass of 35 × 103 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inhibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Furthermore, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts.