The present study was undertaken to determine the protective effects of Melatonin and Vitamin C in combination against renal I/R injury in rats. 40 wistar albino female rats were divided into five groups: Sham, I/R, I/R+Mel, I/R+VitC and I/R+Mel+VitC. While nothing was done in left kidney in Sham group, all I/R groups were subjected to I/R procedure (45 min occlusion and 60 min reperfusion). In I/R +Mel group, all rats were injected by Melatonin 50 mg/kg i.p. for 3 days plus morning of operation day. In I/R+VitC group, all rats were injected by Vitamin C 500 mg/kg/day i.p. 24 hours before I/R procedure. In I/R+Mel+VitC group, all rats were injected by Melatonin and Vitamin C by the same dose and intervals as in I/R+Mel and I/R+VitC groups. After 3 days, rats were exposed to I/R procedure following right nephrectomy. After sacrification, blood and left kidney parts were subjected to biochemical and histopathological examinations. The I/R significantly increased serum urea, creatinine and IMA levels and renal MPO activity and MDA levels, and decreased renal SOD and CAT activities. Pretreatment with Melatonin and Vitamin C markedly reversed these findings. In histology, while cortical necrotic changes of the epithelium lining tubules and glomeruli as well as asymmetrical and shrunken glomeruli were observed in IR group, there was a marked regression of the histopathological changes in combined pretreatment group. In conclusion, combined pretreatment with Melatonin and Vitamin C seems to have higher anti-inflammatory and antioxidant properties than individual pretreatments with either agent alone.