The aim of this study is to determine the protective effects of vitamin D3 and dehydroascorbic acid (DHA), a blood-brain barrier transportable form of vitamin C, against ischemia/reperfusion (I/R) injury on a middle cerebral artery occlusion/reperfusion model of brain since reactive oxygen species play an important role in the pathophysiology of I/R injury in brain. In order to examine antioxidant status and lipid peroxidation, we assayed malondialdehyde (MDA) levels as a marker of lipid peroxidation, and reduced glutathione (GSH) and superoxide dismutase (SOD) enzyme activities as free radical scavenging enzymes in cortex and corpus striatum (CS). Wistar albino rats were divided into five equal groups of each consisting of seven rats: control, I/R, I/R + DHA, I/R + vitamin D3, and I/R + vitamin D3 + dehydroascorbic acid groups. MDA levels were found to be increased in the I/R group, I/R + DHA, and I/R + vitamin D3 groups compared with the control group in both cortex and corpus striatum. However, MDA level were found to be significantly decreased in only I/R + vitamin D3 + DHA group compared with the I/R group in cortex (P < 0.0001). MDA levels were not significantly different in I/R + DHA, and I/R + vitamin D3 groups compared with the I/R group. GSH and SOD enzyme activities were significantly decreased in I/R, I/R + DHA, and I/R + vitamin D3 groups compared with the control group in both cortex and corpus striatum (CS) (P < 0.0001). Whereas, both GSH and SOD activity were increased in I/R + vitamin D3 + DHA group compared with the I/R group in both cortex and CS (P < 0.001 in cortex, P < 0.001 in CS for SOD P < 0.002 in cortex P < 0.03 in CS for GSH). Our results demonstrate that the combination of vitamin D3 and DHA treatment prevent free radical production and dietary supplementation of vitamin D3 and DHA which may be useful in the ischemic cerebral vascular diseases.