BACKGROUND This study aimed to analyze the preventive effect of nitric oxide (NO)-releasing nanofibers against ischemia-reperfusion injury (IRI) and to determine the mechanism of action as a novel NO delivery system in a rat model. MATERIAL AND METHODS Eight-week-old male Sprague-Dawley rats, weighing 250 to 280 g, were divided into 3 groups: sham, untreated (n=5); control, renal ischemia injury for 55 min (n=4); and NO24, renal ischemia injury for 55 min with kidney wrapping of NO-releasing nanofiber for 24 h (n=6). mRNA expression was measured by real-time polymerase chain reaction (PCR), whereas protein expression was assessed by immunohistochemistry and western blot analysis. RESULTS Serum creatinine levels in the sham, control, and NO24 groups were 0.48±0.08, 4.66±0.33, and 2.60±1.00 mg/dL, respectively (P=0.002). Anti-apoptotic Bcl-2 protein expression differed significantly between the control and the NO24 groups (Bcl-2/ß-actin; control, 0.50±0.12; NO24, 1.56±0.56; P=0.024). mRNA expression level of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was significantly higher in the control group (23.24±11.32, P=0.016) than in the sham group (1.00±1.21), and mRNA expression of TNF-alpha in the NO24 group (1.28±1.16, P=0.010) was significantly lower than in the control group. Histological analysis revealed decreased atrophy and necrosis in the NO24 group compared to those in the control group. CONCLUSIONS This study demonstrated that kidney wrapping of NO-releasing nanofibers had a protective effect against kidney IRI through anti-apoptotic and anti-inflammatory mechanisms.