C-terminal Src kinase (Csk)-binding protein (Cbp) is a transmembrane adaptor protein that localizes exclusively in lipid rafts, where it regulates Src family kinase (SFK) activities through recruitment of Csk. Although SFKs are well known for their involvement in cancer, the function of Cbp in carcinogenesis remains largely unknown. In this study, we reported overexpression of Cbp in more than 70% of renal cell carcinoma (RCC) specimens and in the majority of tested RCC cell lines. Depletion of Cbp in RCC cells by RNA interference led to remarkable inhibition of cell proliferation, migration, anchorage-independent growth as well as tumorigenicity in nude mice. Strikingly, silencing of Cbp negatively affected the sustaining of Erk1/2 activation but not c-Src activation induced by serum. Besides, the RhoA activity in RCC cells was remarkably impaired when Cbp was knocked down. Overexpression of wild-type Cbp, but not its mutant Cbp/ΔCP lacking C-terminal PDZ-binding motif, significantly enhanced RhoA activation and cell migration of RCC cells. These results provided new insights into the function of Cbp in modulating RhoA activation, by which Cbp might contribute to renal cell carcinogenesis. © 2009 Macmillan Publishers Limited.