The aim of the present study was to investigate the antiproliferative effects of interferon (IFN)-alpha and rapamycin (RPM) on renal cell carcinoma (RCC) cells and examine the synergistic growth suppression conferred by IFN-alpha and RPM. The effects of IFN-alpha and/or RPM on RCC cells were determined using a WST-1 assay and the synergy of IFN-alpha and RPM against three RCC cell lines was analyzed with isobolographic analysis. The expression of mammalian target of rapamycin (mTOR) was downregulated by RNAi, and the expression and phosphorylation of proteins in the mTOR pathway following treatment with IFN-alpha and/or RPM was examined by western blot analysis. The observations indicated that IFN-alpha significantly increased the susceptibility of RCC cells to RPM and the synergistic effect of IFN-alpha and RPM against RCC cells was confirmed in all three RCC cell lines. The mTOR pathway was shown to be associated with the synergistic effect of IFN-alpha and RPM against RCC. IFN-alpha and RPM alone decreased the phosphorylation of mTOR, p70 S6 kinase, S6 and 4E binding protein 1, and IFN-alpha significantly enhanced the RPM-induced suppression of the mTOR pathway. However, in RCC cells with low mTOR activity, the synergy of IFN-alpha and RPM was eliminated. Therefore, the results of the present study indicate that the mTOR pathway plays an important role in the synergistic effect of IFN-alpha and RPM against RCC cells. Thus, mTOR may serve as an effective therapeutic target in the treatment of advanced RCC.