Serine protease is considered a relative upstream regulator in the physiological processes including male reproduction. Transmembrane serine protease 12 (TMPRSS12) has been shown to regulate sperm motility and uterotubal junction migration in mice, but its role in the testis remains unknown.In this study, we confirmed the important role of TMPRSS12 in spermatogenesis. To further investigate the mechanism of TMPRSS12 in male reproduction, we used 2-D electrophoresis combined with Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry(MALDI-TOF/TOF) to construct differential protein expression profiles of testes in Tmprss12-/- and WT mice. A total of 64 spots with 2-fold or more differential expression between two groups were identified using MALDI-TOF/TOF. As a result, 54 unique proteins were identified successfully, including 39 proteins with decreased expression and 15 proteins with increased expression in Tmprss12-/- mice.