Renal ischemia/reperfusion(I/R) is an important injury part of ischemic acute renal failure, and it is also the main factor that affects the early functional recovery and the long-term survival of transplanted kidney in renal transplantation. In this study, we cloned and expressed truncated Na+/K+-ATPase β(tNKAβ) and demonstrated that tNKAβ could activate NKA α subunit and induce protective effect on human kidney-2(HK-2) cells via PKCɛ signal pathway. The half maximum effective concentrations (EC₅₀) of tNKAβ were 0.24 µM. Furthermore, the application of EAVSLKPT (PKCɛ inhibitor) could abolish the protective effect of tNKAβ in HK-2 cells subjected to ischemia/reperfusion. To identify the protective effect of tNKAβ against the I/R injury in the kidney, Sprague-Dawley rats were treated with tNKAβ (75 mg/kg) for 2 h before ischemia. The tNKAβ-treated group demonstrated a significant improvement in renal function with a lower serum creatinine and blood urea nitrogen (BUN) levels on postoperative days 1-6. Renal sections obtained from rats of the I/R group showed serious renal injury which included degeneration of tubular structure, tubular dilation, swelling and necrosis, luminal congestion, and muddy brown casts formed by sloughing of severely damaged tubular epithelial cells. However, sections of rats that were administered with tNKAβ 2 h before reperfusion showed marked reduction of the histological features of renal injury compared with kidneys that were subjected to I/R only. In conclusion, the protective effects of tNKAβ against renal I/R injury have been evaluated for the first time, and these protective effects may occur via stimulation of PKCɛ pathways.