Total proteins expressed in binucleate stage anthers of Honglian rice CMS line during the development of microspores were separated by two-dimensional electrophoresis, through coomassie blue staining, two dimensional gel electrophoresis profiles with high resolution and reproducibility were obtained. Twelve protein spots were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI–TOF/MS) and 7 were identified by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Among these protein spots, 5 spots were involved in biosynthesis or metabolism of carbonhydroxydate, 3 spots involved in biosynthesis, 5 spots involved in transcription, 2 spots involved in biosynthesis of plant secondary metabolites, 2 spots involved signal transduction, 2 spots involved cell death/disease, and 5 spots with unknown function. Among these 19 protein spots, the expression of 18 spots were down-regulated or absent in the male-sterile line, only one spot, standing for a proteasome subunit (spot 9) was regularly expressed, indicating the abnormal accumulation of carbonhydroxydates and proteins in male-sterile line anthers during the development of pollens might relate to pollen sterility.