The purpose of this research was to evaluate the protective effects of insulin-loaded poly(ethylene glycol)-b-poly((2-aminoethyl-l-glutamate)-g-poly(l-lysine)) (PEG-b-P(ELG-g-PLL)) on renal ischemia/reperfusion (I/R) injury in rats with diabetes mellitus. Rats were preconditioned with free insulin or insulin/PEG-b-P(ELG-g-PLL) polyplexes, then subjected to renal I/R. The blood and kidneys were then harvested, Glucose uptake rate, glucose transporter 4 (GULT4) mRNA level, cell membrane GULT4 content and GULT4 expression were measured, the level of serum creatinine and blood urea nitrogen were determined, the activity of superoxide dismutase and inducible nitric oxide synthase, the content of malondialdehyde and nitric oxide, reactive oxygen species (ROS) production and nuclear factor κB (NF-κB) mRNA level, Bcl-2 assaciated x protein (Bax) mRNA and B cell lymphoma/lewkmia-2 (Bcl-2) mRNA level, and the expression of protein 47kDa phagocyte oxidase (p47phox) in renal tissues were measured. Insulin preconditioning improved the recovery of renal function, reduced oxidative stress injury, restored nitroso-redox balance and downregulated the expression of p47phox induced by renal I/R injury, while the application of block copolymer PEG-b-P(ELG-g-PLL) as an insulin nanocarrier significantly enhanced the protective effect of insulin. Block copolymer PEG-b-P(ELG-g-PLL) could be used as a potential nanocarrier for insulin with sustained release and enhanced bioavailability.