Background: The mechanism of proteinuria observed with rapamycin (RPM) use remains unclear. The conversion from calcineurin inhibitors (CNIs) to RPM in kidney transplant recipients has been associated with a higher incidence of proteinuria. In this study, we performed proteomic analysis to investigate the alteration of protein expression in mouse podocyte treated with RPM in comparison with RPM/CNI combination. Methods: Immotalized mouse podocytes were treated with 20 nM RPM or 20 nM RPM+1 μg/ml cyclosporine. Podocyte proteins were separated by two dimensional-polyacrylamide gel electrophoresis (PAGE) and identified by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and peptide fingerprinting. Selected proteins were analyzed by western blot assay. Results: We identified 36 differentially expressed proteins after isolated RPM or RPM/CNI combination treatment in cultured mouse podocytes. There are three distinct patterns of protein expression: 1. Potentiated down-regulation of proteins by RPM/CNI treatment compared with isolated RPM treatment (n=4); 2. Partial offset of down-regulation by RPM/CNI in comparison with RPM (n=25); 3. No difference in down-regulation between RPM and RPM/CNI (n=7) Conclusion: We found a significant interplay between RPM and CNI on the proteins expression in mouse podocyte. This might explain the higher incidence of proteinuria by RPM/CNI combination in clinical settings. Further study is required to elucidate the target protein associated with RPM induced proteinuria.