Corynebacterium glutamicum JHI3-156 is an L-isoleucine producing strain, but its metabolic mechanism is not fully understood. In this work, total proteins extracted from JHI3-156 and the wild type Corynebacterium glutamicum ATCC 13869 were comparatively analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. About 750 protein spots were visualized on two-dimensional gels from both samples. Compared with the wild type ATCC13869, 82 proteins were up-regulated and 123 proteins were down-regulated in JHI3-156. There were 181 proteins observed only in ATCC13869 and 197 proteins observed only in JHI3-156. 13 protein spots from the ATCC13869 gel and 18 protein spots from the JHI3-156 gel were chosen and further analyzed by MALDI-TOF mass spectrometry. These proteins are involved in cell growth, L-isoleucine biosynthesis and stress response. This study provided some clues to understand the molecular and metabolic mechanisms that lead to the increased L-isoleucine production in Corynebacterium glutamicum.