Hepatocellular carcinoma (HCC) is a frequent malignant tumor. Catalpol is a Chinese medicine extract with a number of pharmacologically active properties. The present study aimed to investigate the effects and mechanisms of catalpol in HCC. HCC cells were treated with catalpol in the presence or absence of microRNA (miR)‑140‑5p inhibitor, and assays to determine cell viability, proliferation, invasion and migration were performed. Reverse transcription‑quantitative PCR and western blotting were performed to determine the mRNA and protein expression levels of miR‑140‑5p, vimentin, N‑Cadherin and E‑Cadherin. Moreover, cells were treated with catalpol in the absence or presence of transforming growth factor (TGF)‑β1, and the cell morphology was observed under a microscope. The results demonstrated that catalpol inhibited cell proliferation, invasion and migration, and decreased the expression levels of vimentin and N‑cadherin, but increased the expression levels of E‑cadherin and miR‑140‑5p. Catalpol inhibited morphological changes in epithelial‑mesenchymal transformation (EMT) of cells induced by TGF‑β1. Following inhibition of miR‑140‑5p expression, the proliferation, invasion and migration of HCC cells were promoted, E‑cadherin expression was decreased, and the levels of vimentin and N‑cadherin were increased. The miR‑140‑5p inhibitor effectively reversed the inhibitory effect of catalpol on cell proliferation, invasion and migration. Thus, the results suggested that the antitumor potential of catalpol in HCC may be exerted by regulating the expression of miR‑140‑5p to inhibit proliferation, invasion, migration and EMT of HCC cells.