The tumor suppressor p53 is known to be able to trigger apoptosis in response to DNA damage, oncogene activation, and certain chemotherapeutic drugs. In addition to its transcriptional activation, a fraction of p53 translocates to mitochondria at the very early stage of apoptosis, which eventually contributes to the loss of mitochondrial membrane potential, generation of reactive oxygen species (ROS), cytochrome c release, and caspase activation. However, the mitochondrial events that affect p53 translocation are still unclear. Since mitochondrial uncoupling has been suggested to contribute to cancer development, herein, we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by mitochondrial uncoupling using chemical protonophores, and further verified the results using a siRNA approach in murine skin epidermal JB6 cells. Our results showed that mitochondrial uncoupling blocked p53 mitochondrial translocation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter to induce p53-mediated apoptosis in skin carcinogenesis. This blocking effect, in turn, led to preservation of mitochondrial functions, and eventually suppression of caspase activity and apoptosis. Moreover, uncoupling protein 2 (UCP2), a potential suppressor of ROS in mitochondria, is important for TPA-induced cell transformation in JB6 cells. UCP2 knock down cells showed enhanced p53 mitochondrial translocation, and were less prone to form colonies in soft agar after TPA treatment. Altogether, our data suggest that mitochondrial uncoupling may serve as an important regulator of p53 mitochondrial translocation and p53-mediated apoptosis during early tumor promotion. Therefore, targeting mitochondrial uncoupling may be considered as a novel treatment strategy for cancer.