Background: Hepatocellular carcinoma (HCC) is the most common type of liver malignancies. Yet, the outcome of the traditional chemotherapeutic agents in the management of HCC still unsatisfactory, most probably due to their limited therapeutic efficacies. Thus, there is an urgent medical need for alternative therapeutic approaches for fighting HCC. Aim: our study aimed at evaluating the possible synergistic antitumor activity of the herbal alkaloid berberine (BER) with the conventional doxorubicin (DOX) in a DENA-induced HCC rat model. Methods: HCC was induced in male Wistar rats by oral DENA administration in their drinking water (100 mg/L) for 8 weeks. In addition to a positive and negative control groups, a group of animals (10 rats) was given DOX (4× 2.5 mg/kg, i.v., weekly). Another group (10 rats) was given BER (100 mg/kg) by oral gavages daily for one month. A third group (10 rats) was given a combination of both BER and DOX in the above described dose and schedule. Results: Combination between DOX and BER was superior over their corresponding individual administrations as indicated by significant improvement in the overall estimated indices of liver function. Also, in comparison to individual therapy, this combination was obviously more potent to reduce the levels of novel HCC related tumor markers including serum AFP-L3 and tissue levels of Golgi protein 73, and glypican 3. Furthermore, histological investigations and assessments of hepatic tissue levels of some oxidative stress markers strongly confirmed the advantageous effects of combined BER and DOX in fighting DENA-induced HCC. Conclusion: our study conclusively revealed that combining BER with DOX exhibited a promising preclinical anticancer efficacy and could be considered as a novel strategy to synergistically combat HCC in clinical practices.