Background: Withaferin A is a triterpenoid steroidal lactone and is commonly isolated from Withania somnifera. It has been shown to have tremendous pharmacological potential. Objectives: The present study was aimed at elucidating the mechanism of action of withaferin A against hepatocellular carcinoma (HCC) cells via the PI3K/AKT signaling pathway. Materials and Methods: MTT (tetrazolium dye) Assay was performed for the relative assessment of the viabilities of cell lines. The effect of withaferin A on the proliferative capability of HCC cells was analyzed using the EdU staining assay, and the colony-forming potential was assessed with the help of a clonogenic assay. Cell apoptosis was estimated through (Modified Annexin V/Propidium Iodide Apoptosis Assay) staining followed by flow cytometry. Transwell assays were carried out for estimating the migration and invasion of cancer cells. The qRT-PCR and western blotting, respectively, were used for gene and protein expression studies. Results: Withaferin A selectively inhibited the viability of HCC cells although the viability of normal liver cells was minimally affected. The IC50 of withaferin A against the HepG2 cells was found to be 12 μM as against 150 μM for normal THLE-2 cells. The treatment with withaferin A significantly minimized the proliferation and colony-forming potential of cancer cells by inducing cell apoptosis. The percentage of apoptosis increased from 3.8% in control to 20.3% at 24 μM withaferin A. The cancer cell migration and invasion were significantly declined by withaferin A together with the inhibition of Epithelial to mesenchymal transition (EMT) of HCC cells. The withaferin A treatment decreased the expression of carcinoma cell markers CD44, CD90, and EpCAM. The expression of miR-200c markedly increased under withaferin A treatment and the latter was shown to exert its anti-cancer effects through miR-200c-mediated inhibition of the PI3K/AKT signaling pathway in HCC cells. Conclusion: In conclusion, withaferin A modulated the expression of miR-200c in HCC cells to inhibit the PI3K/AKT pathway and restricted the cancer cell EMT together with the inhibition of in vitro cancer cell growth and viability via induction of apoptosis.