Abstract: Background and Objective: Antineoplastic acts by numerous mechanisms and have variability in action on healthy and cancerous cells. To examine the protective effect of Glucose-6-Phosphate Dehydrogenase (G6PD) and Dihydrofolate Reductase (DHFR) in Diethylnitrosamine (DENA) induced hepatocellular carcinoma in rats. Materials and Methods: A total of 30 healthy male divided into 5 groups (n = 6): Group 1 rats entitled normal controls, rats of Group 2 was serving as disease controls and exposed to a single dose of DENA (200 mg kg–1, IP). Group 3, 4 and 5 were treatment groups that were subjected to DENA administration as scheduled in group-2 and treated with primaquine (PQ) (0.21 mg kg–1 per day, administered p.o.), methotrexate (MTX) (7.5 mg kg–1 per 3 doses per week) and low dose PQ+MTX (0.12 mg kg–1 per day p.o.+7.5 mg kg–1 per 3 doses per week) respectively for three weeks. The serum Aspartate Transaminase (AST), Lactate Dehydrogenase (LDH), Alanine Aminotransferase (ALT), α-fetoprotein levels were estimated. In liver tissue, levels of Catalase (CAT), Glutathione (GSH) and Malondialdehyde (MDA) were estimated. The levels of NF-κB, Bcl-2 and IkB-α were measured using western blot. Results: Serum levels of AST, ALT, LDH and α-fetoprotein elevated significantly in group 2 animals which were found maintained in treatment animals. The levels of antioxidant enzymes level of NF-κB, Bcl-2 and I κB-α were also altered in disease control group animals which were restored in treated animals. Results of group 5 were more consistent and satisfactory. Conclusion: The lower dose combination therapy with an inhibitor of G6PD and DHFR can successfully manage toxicities associated with methotrexate and may reduce the dose of methotrexate to a safer level.