Tumor cells can adapt to a hostile environment with reduced oxygen supply. The present study aimed to identify mechanisms that confer hypoxia resistance. Partially hypoxia/reoxygenation (H/R)-resistant proximal tubular (PT) cells were selected by exposing PT cultures to repetitive cycles of H/R. Thereafter, H/R-induced changes in mRNA and protein expression, inner mitochondrial membrane potential (ΔΨ(m)), formation of superoxide, and cell death were compared between H/R-adapted and control PT cultures. As a result, H/R-adapted PT cells exhibited lower H/R-induced hyperpolarization of ΔΨ(m) and produced less superoxide than the control cultures. Consequently, H/R triggered ΔΨ(m) break-down and DNA degradation in a lower percentage of H/R-adapted than control PT cells. Moreover, H/R induced upregulation of mitochondrial uncoupling protein-3 (UCP-3) in H/R-adapted PT but not in control cultures. In addition, ionizing radiation killed a lower percentage of H/R-adapted as compared to control cells suggestive of an H/R-radiation cross-resistance developed by the selection procedure. Knockdown of UCP-3 decreased H/R- and radioresitance of the H/R-adapted cells. Finally, UCP-3 protein abundance of PT-derived clear cell renal cell carcinoma and normal renal tissue was compared in human specimens indicating upregulation of UCP-3 during tumor development. Combined, our data suggest functional significance of UCP-3 for H/R resistance.