Background: The exploration of bacterial endospores as potential antitumor agents has garnered increased attention in recent years. This study investigates the impact of gamma radiation-attenuated Clostridium sporogenes endospores as modifiers of immune regulation on tumor growth, with the aim of refining therapeutic protocols for hepatocellular carcinoma. Methods: Hepatocellular carcinoma was induced in adult male albino rats using diethylnitrosamine (DEN) (20mg/kg b.wt/day) orally injected five times a week for 6 consecutive weeks) and subsequently treated with endospores (intravenous injection of endospores  once at a dose of (60 million of spores /rat) and samples were taken from rat after one month of endospore injection). Multiple parameters, including interferon gamma (IFN-γ), nuclear factor-κB (NF-κB), tumour necrosis factor alpha (TNF-α), antioxidant enzymes, malondialdehyde (MDA), and liver function enzymes were assessed. Results: Results demonstrated a remarkable suppression of tumor proliferation coupled with the induction of enduring immunity through IFN-γ stimulation. The levels of inflammatory markers associated with tumor promotion (NF-κB and TNF-α) witnessed significant improvement. This was paralleled by enhancements in antioxidant status and a reduction in lipid peroxidation in liver tissues of the endospore-treated rats compared to the untreated rats. Histopathological examinations, along with elevated liver function enzymes, provided additional support for these observed improvements. Conclusion: This study offers compelling evidence for the potential of endospores in modulating immune responses and targeting tumor cells, suggesting their plausible roles as adjuvants to augment cancer therapeutic protocols