To identify new biomarkers that facilitate the accurate early diagnosis of osteosarcoma and that may possibly include novel therapeutic candidates, we performed a proteomic approach to compare osteosarcoma cells and human primary cultured osteoblastic cells. Image analysis of silver-stained 2-DE gels revealed that the level of 12 protein spots was significantly different between the two groups of samples (p < .004). After mass spectroscopic identification and database searches, we found that in osteosarcoma cells, the level of HSP70, actin capping protein, ATP synthase, Mthsp75, UQCRC1, Ras-related nuclear protein, UCH-L1, and PRDX4 was elevated. However, the level of pyruvate dehydrogenase E1, Prohibitin, and Annexin V was decreased. Subsequent Western blot analyses of UQCRC1, UCH-L1, and PRDX4 in osteosarcoma tissues confirmed the results obtained by the proteomic analyses. These identified proteins may be potential molecular targets for osteosarcomatous diagnostics and therapeutics.