Background: Hepatocellular carcinoma is on the rise globally. The anticancer effects of metformin have been mainly studied in the context of supra-pharmacological concentrations (>1 mM). In this study, we aim to investigate the effects of therapeutic doses of metformin on the lncRNA-AF085935/glypican-3/mitogen-activated protein kinase pathway in HCC. Methods: HepG2 cells were treated with various concentrations of metformin (0, 5, 10 and 20μM) for different durations (24, 48, and 72h). Cell viability was evaluated by MTT assay, while gene expression levels of lncRNA-AF085935, glypican-3, VEGF, P53, NFκB genes were evaluated by quantitative real-time PCR. The ratio between the phosphorylated p44/42 and the total p44/42 protein level was assessed by western blot. Results: Compared to the control groups, we found a significant decrease in cell proliferation, lncRNA-AF085935 gene expression (p value <0.001), VEGF gene expression (p value <0.001) and p-p44MAPK protein level (p value <0.0001) in all metformin-treated groups except the metformin 5µM-24h group. Furthermore, our data showed a significant decrease in gene expression for both glypican-3 and NFκB at 48 and 72h compared to the control group (p value <0.001, 0.001) respectively, with no significant effect of metformin after 24h. Also, we found a significant increase in p53 gene expression in 20μM group compared to the corresponding control cells of the same duration, while the 5μM group showed no significant difference in any duration. Conclusion: Our research gives new insights into metformin's anti-carcinogenic effect on HCC through its impact on LncRNA-AF085935/GPC3/MAPK pathway.