Objective To explore the effect and mechanism of intraoperative remifentanil (RF) on renal ischemia/reperfusion (I/R) injury. Methods Fifty male C57/BL mice were randomly divided into 5 groups:sham group, I/R group, I/R+LY294002 (a phosphatidylinositol 3-kinase[PI3K] inhibitor) group, I/R+RF group and I/R+RF+LY294002 group, with 10 mice in each group. Venous blood or renal tissue samples were collected from the mice of each group 6 h after I/R operation. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected using automatic biochemical analyzer. The expression levels of PI3K/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway-related proteins in renal tissues of mice were detected using Western blotting. The aggregation of inflammatory cells was observed by H-E staining. The expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and IL-10 in renal tissues of mice were detected by enzyme-linked immunosorbent assay. The mRNA expression levels of anti-apoptotic factor Bcl2 and apoptotic factor Caspase-3 in renal tissues were determined by real-time quantitative PCR. Results Compared with the sham group, the BUN and SCr levels in venous blood were increased in the I/R group, the PI3K expression, phosphorylated-Akt (p-Akt)/Akt ratio and phosphorylated-eNOS (p-eNOS)/eNOS ratio in renal tissues were decreased, the release levels of inflammatory factors (TNF-α, IL-1β, IL-6 and IL-10) were increased, Bcl2 mRNA expression was decreased, and Caspase-3 mRNA expression was increased; and the differences were significant (all P<0.05). The mice of the I/R group had increased inflammatory cell recruitment in renal tissues. After RF treatment, the mice of the I/R+RF group had decreased levels of BUN and SCr in venous blood, increased PI3K expression, p-Akt/Akt ratio and p-eNOS/eNOS ratio in renal tissues, decreased release levels of inflammatory factors (TNF-α, IL-1β, IL-6 and IL-10), increased Bcl2 mRNA expression, and decreased Caspase-3 mRNA expression; and the differences were significant compared with the mice of the I/R group (all P<0.05). The inflammatory cell recruitment was decreased in the I/R+RF group. Moreover, compared with the mice of the I/R+RF group, the mice of the I/R+RF+LY294002 group had increased levels of BUN and SCr in venous blood, decreased p-eNOS/eNOS ratio in renal tissues, increased IL-1β and IL-6 release, and increased Caspase-3 mRNA expression; and the differences were significant (all P<0.05). The inflammatory cell recruitment was increased in the I/R+RF+LY294002 group. Conclusion RF exerts protective effect on kidney with I/R injury by alleviating renal inflammation and cell apoptosis through activating PI3K/Akt/eNOS pathway.