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Currently, the outcomes of conventional chemotherapeutic approaches are unsatisfactory. Clinical application of nanoparticles seems promising. We aim to evaluate the possible antitumor activity of zinc oxide nanoparticles (ZnONPs) as a chemotherapeutic approach in in vitro and in vivo experimental models. An in vitro study was performed on three different cell lines, namely human hepatocellular carcinoma (HEPG2), human prostate cancer (PC3), and none-small cell lung cancer (A549) cell lines. An in vivo study using diethylnitrosamine (DENA)-induced HCC in adult male Wistar rats was conducted to investigate the potential antitumor activity of ZnONPs in HCC and the possible underlying mechanisms. Hepatocellular carcinoma (HCC) was induced by oral administration of DENA given in drinking water (100 mg/L) for 8 weeks. Rats were allocated into four groups, namely a control group, an HCC control group receiving DENA alone, a ZnONPs (10 μg/kg per week, intravenous (i.v.) for 1 month) control group, and a ZnONPs treatment group (receiving ZnONPs + DENA). ZnONPs significantly reduced the elevated serum levels of HCC-related tumor markers alphafetoprotein and alpha-l-fucosidase and the apoptotic marker caspase-3 compared with the untreated HCC rats. In addition, treatment with ZnONPs significantly decreased the elevated levels of hepatocyte integrity and oxidative stress markers as compared with the untreated HCC control group. Furthermore, the histopathological study revealed anaplasia and fibrous degenerations which were significantly corrected by ZnONPs treatment. In conclusion, administration of ZnONPs exhibited a promising preclinical anticancer efficacy in HCC and could be considered as a novel strategy for the treatment HCC in clinical practices.