Probiotics have been suggested as a safe and cost-effective approach to prevent or treat hepatocellular carcinoma (HCC). Some of the exopolysaccharides (EPSs) produced by lactic acid bacteria confer health benefits such as immunomodulatory and antitumor activities. The present study was therefore aimed to investigate the immunomodulatory effect of Lactobacillus acidophilus ATCC 4356 EPSs against diethylnitrosamine (DEN) and gamma radiation (IR) induced HCC either as prevention or treatment in male rats' model. Biochemical results revealed a significant increase in serum ALT and γ-GT activities as well as MDA, IL-17, TGF-β1, signal transducer and activator of transcription-3 protein (STAT3), mitogen-activated protein kinase p38 (p38MAPK) levels in the liver tissue. The gene expression level of the liver toll-like receptor 2 (TLR-2) gene was also increased. However, prevention and treatment with EPSs ameliorated most of the investigated parameters. The histopathological observations of liver tissues were in agreement with restored biochemical results. In conclusion, Lactobacillus acidophilus ATCC 4356 EPSs are efficacious control against HCC throughout the regulation of TLR2/STAT-3/P38-MAPK Pathway associated with inflammation. Therefore, our novel EPSs ATCC 4356 could be used as a good, safe and effective probiotic to prevent hepatocarcinogenesis in suspected patients.