Objective To observe the effect of single intensive hyperbaric oxygenation (HBO) on cytochrome C and caspase-3 in rats after permanent middle cerebral artery occlusion (MCAO) very early. Methods Forty-eight male Sprague-Dawley rats were subjected to permanent MCAO model using the intraluminal suture method, and were divided into control group (n=24) and HBO group (n=24). The HBO group stayed in the hyperbaric cabin with a pressure of 0.2 MPa for 9 hours 3 hours after MCAO. They were measured with Garcia scores 3 hours, 13 hours and 24 hours after MCAO. Apoptosis cells of ischemic penumbra tissue were investigated with TUNEL 13 hours and 24 hours after MCAO, while the level of cytochrome C and caspase-3 were measured with ELISA. Results The Garcia scores increased 13 hours and 24 hours after MCAO in both groups, but there was no significant difference between groups (t<2.07, P>0.05). The apoptosis cells were found in both groups 13 hours and 24 hours after MCAO, and less in the HBO group than in the control group (t>6.57, P<0.01). The levels of cytochrome C and caspase-3 were less in the HBO group than in the control group 24 hours after MCAO (t>2.41, P<0.05). Conclusion A single intensive HBO in very early stage may improve neurological function after cerebral ischemia in rats, which may associate with the inhibition of cytochrome C and caspase-3 to reduce cell apoptosis.