Renal ischemia/reperfusion (I/R) is an alternation of renal hemodynamics, which results in diverse postischemic responses and eventually acute kidney injury. Although renal ischemic preconditioning (IPC) is known to protect the kidney from I/R injury, the precise renoprotective mechanisms are not completely understood. The multiple renoprotective effects of IPC underscore the importance in understanding molecular mechanisms and the targets of action involved. This study aimed to identify the biochemical changes in renal I/R injury and investigate the renoprotective mechanisms of IPC. Herein, renal I/R was produced in adult male Sprague–Dawley rats through the bilateral ligation of renal pedicles for 45 min, followed by reperfusion for 24 h. For the IPC group, rats were subjected to three cycles of 2‐min ischemia, followed by a 5‐min reperfusion, 15 min prior to renal I/R. Our data confirmed the beneficial effects that IPC has on renal I/R injury. IPC‐mediated renoprotection was associated with the resolution of oxidative stress, inflammation, apoptosis, and hyperglycemia. Among the numerous signaling molecules involved in the renoprotective mechanisms of IPC, an elevated protein expression of Nrf2, HO‐1, LC3 II conversion, along with Atg12 and protein phosphorylation of AMPK, as well as a decreased protein phosphorylation of ERK, p38 MAPK, and Akt and NF‐κB DNA binding activity were identified. Importantly, the post renal I/R overproduction of counter‐regulatory hormones, impaired hepatic insulin action, and augmented hepatic gluconeogenesis were improved through IPC. As counter‐regulatory hormones have been implicated in the induction of oxidative stress, inflammation, apoptosis, impaired insulin action, hyperglycemia, and tissue destruction, our findings suggest that counter‐regulatory hormones may well be valuable targets of IPC for combatting renal I/R injury. © 2018 IUBMB Life, 71(3):321–329, 2019