The immotile testicular mammalian spermatozoon gets transformed into a motile spermatozoon during 'epididymal maturation'. During this process, the spermatozoa transit from the caput to the cauda epididymis and undergo a number of distinct morphological, biophysical and biochemical changes, including changes in protein composition and protein modifications, which may be relevant to the acquisition of motility potential. The present proteome-based study of the hamster epididymal spermatozoa of caput and cauda led to the identification of 113 proteins spots using Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis. Comparison of these 113 protein spots indicated that 30 protein spots (corresponding to 20 proteins) were significantly changed in intensity. Five proteins were increased and eleven were decreased in intensity in the cauda epididymal spermatozoa. In addition, two proteins, glucose-regulated protein precursor (GRP78) and tumor rejection antigen (GP96), were unique to the caput epididymal spermatozoa, while one protein, fibrinogen-like protein 1, was unique to cauda epididymal spermatozoa. A few of the five proteins, which increased in intensity, were related to sperm metabolism and ATP production during epididymal maturation. The changes in intensity of a few proteins such as ERp57, GRP78, GP96, Hsp60, Hsp70, and dihydrolipoamide S-acetyltransferase were validated by immunoblotting. The present study provides a global picture of the changes in protein composition occurring during hamster sperm epididymal maturation, besides being the first ever report on the proteome of hamster spermatozoa.