Tubeimoside　I　(TBMS)　is　a　natural　compound　with　antitumor　properties.　However,　the　detailed　mechanism　underlying　the　function　of　TBMS　in　liver　cancer　has　not　been　fully　elucidated.　In　the　present　study,　TBMS　was　shown　to　suppress　cell　proliferation　and　induce　S　phase　cell　cycle　arrest　in　HepG2　cells.　Furthermore,　TBMS　treatment　induced　autophagy,　evidenced　by　autophagosome　accumulation,　and　increased　the　mRNA　expression　of　Beclin　1　and　microtubule-associated　protein　1　light　chain　3　(LC3)-I.　However,　flow　cytometry　analysis　demonstrated　that　TBMS　exerted　no　effect　on　cell　apoptosis.　Moreover,　TBMS　increased　the　phosphorylation　of　AMP-activated　protein　kinase　(AMPK)　in　a　concentration-dependent　manner,　thereby　activating　the　AMPK　signaling　pathway.　A　specific　AMPK　inhibitor,　compound　C　(CC),　caused　markedly　suppressed　TBMS-induced　accumulation　of　LC3-II.　In　addition,　the　mRNA　expression　of　LC3-Ι　and　Beclin　1　was　also　suppressed　in　cells　treated　with　TBMS　and　CC　in　combination.　The　results　of　the　present　study　provide　new　insights　into　the　role　of　TBMS　in　inducing　autophagy　and　support　the　potential　application　of　TBMS　for　liver　cancer　treatment　in　the　clinical　setting.　©　2020　Spandidos　Publications.　All　rights　reserved.