Aims: Perioperative acute kidney injury (AKI) resulting from renal ischemia reperfusion (IR) is not conducive to the postoperative surgical recovery. Our previous study demonstrated that reactive oxygen species (ROS) transmitted by gap junction (GJ) composed of connexin32 (Cx32) contributed to AKI. However, the precise underlying pathophysiologic mechanisms were largely unknown. The present study focuses on the underlying mechanisms related to ROS transmitted by Cx32. responsible for AKI aggravation. Results: In a set of vivo studies, renal IR was found to cause severe impairment in renal tissues with massive ROS generation, and occurred contemporaneously with activation of NF-κB/p53/PUMA-mediated mitochondrial apoptosis pathways. Cx32 deficiency alleviated renal IR-induced AKI, and simultaneously attenuated ROS generation and distribution in renal tissues, which further inhibited NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathways. Correspondingly, in a set of in vitro studies, hypoxia reoxygenation (HR)-induced cellular injury and cell apoptosis in both human kidney tubular epithelial cells (HK-2) and rat kidney tubular epithelial cells (NRK52E) were significantly attenuated by Cx32 inhibitors or Cx32 gene knock-down. More importantly, Cx32 inhibition not only decreased ROS generation and distribution in human or rat kidney tubular epithelial cells, but also inhibited its downstream NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathway activation.