Hepatocellular carcinoma (HCC) is among the most prevalent and lethal cancers worldwide. Chitosan-coated iron oxide nanocomposite (FeO/Cs) is a promising bio-nanomaterial for many biological applications. The objective of this research was to evaluate the anticancer efficacy of FeO/Cs against HCC in animal models. FeO nanoparticles were prepared and added to chitosan solution; then, the mixture was exposed to gamma radiation at a dose of 20 kGy. Rats have received diethylnitrosamine (DEN) orally at a dose of 20 mg/kg body weight 5 times per week during a period of 10 weeks to induce HCC and then have received FeO/Cs intraperitoneal injection at a dose of 50 mg/kg body weight 3 times per week during a period of 4 weeks. After the last dose of FeO/Cs administration, animals were sacrificed. DEN induced upregulation of PI3K/Akt/mTOR and MAPK (ERK, JNK, P38) signaling pathways and inflammatory markers (TLR4, iNOS, and TNF-α). DEN also decreases cleaved caspase-3 and increases liver enzymes (ALT, AST, and GGT) activities. Administration of FeO/Cs significantly ameliorated the above-mentioned parameters.