Clear cell renal cell carcinoma (ccRCC) is characterized by intratumoral hypoxia and chemoresistance. The hypoxia-inducible factors HIF1α and HIF2α play a crucial role in ccRCC initiation and progression. We previously identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway as a new modulator of HIF1α and HIF2α under hypoxia in various cancer cell models. Here, we report that FTY720, an inhibitor of the S1P signaling pathway, inhibits both HIF1α and HIF2α accumulation in several human cancer cell lines. In a ccRCC heterotopic xenograft model, we show that FTY720 transiently decreases HIF1α and HIF2α intratumoral level and modifies tumor vessel architecture within 5 days of treatment, suggesting a vascular normalization. In mice bearing subcutaneous ccRCC tumor, FTY720 and a gemcitabine-based chemotherapy alone display a limited effect, whereas, in combination, there is a significant effect on tumor size without toxicity. Noteworthy, administration of FTY720 for 5 days before chemotherapy is not associated with a more effective tumor control, suggesting a mode of action mainly independent of the vascular remodeling. In conclusion, these findings demonstrate that FTY720 could successfully sensitize ccRCC to chemotherapy and establish this molecule as a potent therapeutic agent for ccRCC treatment, independently of drug scheduling. Mol Cancer Ther; 15(10); 2465-74. ©2016 AACR.