Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the antiapoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADPribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.