Background: In recent years, a large number of natural compounds have been identified and proved to have a potential cancer chemopreventive importance due to their strong antioxidant and cytotoxic activities. Objective: The present study is designed to investigate the preventive effects of Rutin against N-Nitrosodiethylamine-induced and Phenobarbital-promoted Hepatocellular carcinoma in male wistar rats. Materials and Methods: Twenty-four male wistar rats were divided into four groups (n=6). Group I serve as Control, Group II was induced HCC by DEN (200mg/kg b.wt) followed by phenobarbital (0.05 % w/v), Group III received same as Group II and followed by Rutin (50mg/kg b.wt) and Group IV received Rutin alone at a dose of 50mg/kg b.wt, up to 16 successive weeks. Results: Results observed that there is significant increase in relative liver weight and liver marker enzymes (P<0.001), tumor marker enzymes (AFP, CEA at P<0.001) and also Significant abnormalities were observed in membrane bound enzymes and Electrolytes, whereas the above said alterations were significantly restored in Rutin treated group compared with DEN treated Group. Conclusion: The results of these findings suggested that Rutin can be used as an adjunct to conventional chemopreventive agent, which may provide a novel therapeutic approach to serve as a promising agent for treatment of Hepatocellular carcinoma