Objective To identify the differential proteomic expressions between the liver tissues of male and female mice, and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE, 325 protein spots were differentially expressed (|ratio| ≥ 1.5, P< 0.05) between the liver tissues of male and female mice, in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females, 14 and 34 proteins were up- or down-regulated in males, respectively. Among them, 6 differentially expressed proteins were validated by Western blot which confirmed the reliability of 2D-DIGE results.GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component, molecular function and biological process. 6 pathways were significantly different between the liver tissues of males and females depending on KEGG analysis. Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.