M-like protein was reported mostly in Streptococcus equi and S. suis, and was a kind of important antigen of Streptococcus. In order to analyze antigenicity of M-like protein S. iniae, three different extraction methods (hot acid extraction, alkaline extraction and pepsin digestion) were used for preparing M-like proteins from Streptococcus iniae and Streptococcus dysgalactiae. The M-like proteins of the two streptococci were analyzed by using SDS-PAGE and Western-blotting. By comparing the results of the three methods, the M-like proteins extracted by hot acid had the strongest immunogenicity. The SDS-PAGE diagrams of the M-like proteins of the two streptococci were distinctly different. The M-like proteins extracted from S. iniae contained 7 minor protein bands with molecular weights of 60, 48, 37, 33, 27, 24 and 15 ku, the M-like proteins extracted from S. dysgalactiae contained 2 minor protein bands with molecular weights of 68 and 42 ku. After SDS-PAGE, in the Western-blotting with rabbit antiserum against S. iniae whole cells, only 2 M-like proteins of S. iniae with molecular weights at 60 ku and 37 ku were detected, and only 2 M-like proteins of S. dysgalactiae with molecular weights at 68 ku and 37 ku. It showed that the M-like proteins of the two streptococci had different constitutions and antigenicity.