Trichomoniasis caused by Trichomonas vaginalis is the most common sexual transmitted infection in the world. The 170-MB genome of this protozoan contains 60,000 genes, the largest number of genes ever identified in protozoan. High-throughput expression sequenced tag analysis showed that at least 4,000 genes were expressed in the trophozoite stage. In the present study, we use two-dimensional electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis to profile, identify, and characterize proteins expressed in the trophozoite stage of T. vaginalis. A total of 247 spots representing 164 different proteins were identified. The identified proteins with known sequence or motif/domain homologies were further classified into groups according to their biological functions. Among them, proteins related to carbohydrate metabolism represented the most abundant category in the T. vaginalis proteome. This study presented the most extensive proteomic analysis of T. vaginalis to date and provided a reference proteome database for future comparative proteomic studies.