Abstract: 【Objective】 The present paper was aimed at the comparative proteomic study of anther at binucleate pollen stage between NJCMS1A and its maintainer NJCMS1B in soybean. 【Method】 Two-dimensional gel electrophoresis (2-DE) technique was used to separate the protein spots and Coomassie Blue G-250 was applied to stain the gels. The PDQuest image software was applied to analyze the difference between the protein maps of anthers from NJCMS1A and NJCMS1B. Then the matrix-assisted laser-adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technique was used to obtain the peptide mass fingerprinting of the differentially expressed proteins and the MASCOT software was used to search the protein database NCBInr to identify the spots interested. 【Result】 About 212 protein spots were detected within Mr18.4～116.0 kD and pH 4～7. Total 24 spots out of 212 were differentially expressed between NJCMS1A and NJCMS1B. Among these, 10 protein spots were present in the anther protein map of NJCMS1A but absent in that of NJCMS1B, and 12 protein spots present in that of NJCMS1B but absent in that of NJCMS1A, another two protein spots were up-regulated significantly in the map of NJCMS1B in comparison with that of NJCMS1A. The identified results were as follows: 10 proteins were present in NJCMS1A anther at binucleate pollen stage but absent in NJCMS1B. These were 1-aminocyclopropane-1 -carboxylate oxidase , AIG1-like protein, 20S proteasome beta 5 subunit, Oligouridylate binding protein, Cysteine proteinase, Cullin, Putative beta-amyrin synthase, Hypothetical protein MtrDRAFT_AC146570g8v1, Vacuolar H+-ATPase A subunit, Adenosine/AMP deaminase. Six proteins were absent in NJCMS1A anther but present in NJCMS1B. These were NBS-type resistance protein, putative NBS-LRR protein GS05, MADS box protein, Starch branching enzyme, ACC synthase 2, UDP-glucose pyrophosphorylase.【Conclusion】 According to the literature, the functions, especially those related to male sterility of the major differentially expressed proteins, including 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), ACC synthase 2, cysteine proteinase, vacuolar H+-ATPase A subunit, MADS box protein, starch branching enzyme and UDP-glucose pyrophosphorylase were reviewed and discussed. It was inferred that the cytoplasmic-nuclear male-sterility of NJCMS1A might be related to energy metabolism turbulence, the programmed cell death (PCD), ethylene excessive synthesis, starch synthesis suffocation and the regulation of the flower developmental gene.