AIM: To investigate the chemopreventive effects of 1α,25-dihydroxyvitamin D3 in diethylnitrosamine induced,phenobarbital promoted rat hepatocarcinogenesis.METHODS: The rats were randomly divided into 6 different groups (A-F). Groups A, C and E rats received a single intraperitoneal (i.p) injection of diethylnitrosamine (DEN)at a dose of 200 mg/kg body mass in 9 g/L NaCl solution at 4 wk of age, while group B served as normal vehicle control received normal saline once. After a brief recovery of 2 wk,all the DEN treated rats were given phenobarbital (PB) at 0.5 g/L daily in the basal diet till wk 20. Group A was DEN control. Treatment of 1α, 25-(OH)2D3 in group C was started 4 wk prior to DEN injection and continued thereafter till wk 20 at a dose of 0.3 μg/100 μL propylene glycol per one single dose (os) twice a week. Group E received the treatment of 1α, 25-(OH)2D3 at the same dose mentioned as above for 15 wk. The rats in group D and F received 1α,25-(OH)2D3 alone as in group C without DEN injection.RESULTS: The comet assay showed statistically higher mean values for length to width ratios (L: W) of DNA mass and tailed cells (P＜0.01; P＜0.01 respectively) in DEN treated rats as compared to their normal controls. Continuous supplementation of 1α, 25-dihydroxyvitaminD3 showed a significant (P＜0.01) decrease in L:W ratio of DNA mass tailed cells. Furthermore, 1α, 25-(OH)2D3 supplementations elevated the super oxide dismutase (SOD) activity, hepatic malondialdehyde (MDA) level, reduced glutathione (GSH)and glutathione S-transferase (GST) activity (P＜0.01,P＜0.05, P＜0.05 and P＜0.05 respectively). As an endpoint marker histological changes were observed to establish the chemopreventive effects of 1α, 25-dihydroxyvitaminD3.CONCLUSION: Supplementations of 1α, 25-(OH)2D3 has a marked protection against hepatic nodulogenesis,antioxidant enzymes and DNA damages in DEN induced rat hepatocarcinogenesis promoted by phenobarbital.