The dimensional electrophoresis profiles with good resolution and repeatability was got by separating the proteins of alfalfa on the bud of male sterile (Ms-4) and fertile plants(MF) of bud emergence stage.By using ImageMaster 2D software to analyze silver stained 2-DE gel,the protein spots of Ms-4 and MF mainly scattered in the isoelectric point of 5~7 with maximum distribution of molecular weight in 20~60 kD.There were about 6 000 expressed protein points in bud for Ms-4 and MF,and about 98 spots were differential expressed.The 22 protein spots were successfully identified by mass spectrometry analysis.The ribulose bisphosphate carboxylase small chain,UTP-glucose-1-phosphate uridylyltransferase and others played an important role in photosynthesis,carbohydrate metabolism and the development of multicellular organisms by using Blast2GO program to analyze function and metabolic pathway of the 22 proteins.Meanwhile,they were involved in the composition of cytoplasm,cell wall and other parts,which has some functions,including binding,catalytic,combining and hydrolysis and others.It concluded preliminary that the lack of protein and the changes of expression levels might lead to male sterility,which may attributed to the loss of energy and the change of material synthesis in the development process of alfalfa pollen.