The high recurrence and improved survival rate of clear-cell renal cell carcinoma (ccRCC) demand for continuous effort to search for novel prognostic factors. Herein a comparative proteomics approach based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to identify differentially expressed proteins between ccRCC cell line RLC-310 and normal renal cell line HK-2. Of the 31 proteins identified, Cyclophilin A (CypA) was a newly identified differentially expressed protein in ccRCC cell line. The overexpression of CypA in ccRCC tissues was confirmed using RT-PCR and Western blot analyses. Further immunohistochemistry revealed that overexpression of CypA was associated with poor differentiation and decreased survival (p < 0.05, p < 0.0001). These data suggest that CypA may serve as a novel prognostic factor for ccRCC.