We have previously reported that connexin (Cx) 32 gene, a member of gap junctions, was specifically downregulated in human renal cell carcinoma (RCC) and it acts as a tumor suppressor against RCC. Because there is no standard therapy for advanced RCC, we investigated the anti-metastatic effect of Cx32 to seek a possibility of new RCC therapy. In this study, we used human metastatic RCC cell (Caki-1), and established Cx32-expressed cell clone (Caki-1T) or only mock-transfected cell clone (Caki-1W). For experimental production of metastases, the cells were injected into the lateral tail vein of SCID mice. Seventy days after inoculation, metastatic colonies were observed in Caki-1W inoculated group, though none of them were in Caki-1T inoculated group. The plasma VEGF concentration was significantly lower in Caki-1T group compared to Caki-1W group. To investigate where Cx32 effects on, we also tried in vitro analysis and found that the malignant phenotypes involving metastasis steps were significantly decreased in Caki-1T under hypoxia, a mimic condition of internal tumor environment. After hypoxia treatment, the protein level of HIF-2α, which plays main role for hypoxia adaptation, was observed to increase in Caki-1W, whereas no expression was observed in Caki-1T. We investigated the activation of Src, which is required for stabilization of HIF-2α, is suppressed in Caki-1T compared to Caki-1W. These results suggest that Cx32 inhibits hypoxia adaptation governed by HIF-2α, and this may help to reduce the metastasis of RCC cells. © 2007 Wiley-Liss, Inc.