The　PI3K/Akt　pathway　is　overexpressed　in　nearly　50%　of　hepatocellular　carcinomas　and　inhibits　apoptosis　by　promoting　the　expression　of　antiapoptotic　genes.　Serine　protease　inhibitors　have　been　shown　to　induce　apoptosis　in　hepatoma　cells　by　downregulating　SPINK13　in　the　PI3K/Akt　pathway.　In　this　study,　SPINK13　was　expressed　in　lentiviral　vectors.　Changes　in　signaling　pathway　adapter　proteins,　apoptosis　regulatory　proteins,　cell　cycle　regulatory　proteins,　and　the　biological　behavior　of　hepatocellular　carcinoma　were　observed　in　cell　and　nude　mouse　xenograft　models.　The　underlying　mechanism　of　endogenous　SPINK13-induced　apoptosis　in　hepatocellular　carcinoma　cells　was　explored　via　transcriptomics.　As　a　result,　endogenous　SPINK13　might　inhibit　the　activity　of　Furin　protease,　downregulate　the　Notch1/Hes1　pathway　in　a　binding　manner,　activate　the　direct　effector　PTEN,　inhibit　Akt　phosphorylation,　inactivate　the　downstream　PI3K/Akt　pathway,　and　ultimately　lead　to　mitochondrial　apoptosis　and　cell　cycle　arrest　in　hepatoma　cells.　Therefore,　the　Notch1/Hes1/PTEN　pathway　may　act　upstream　of　SPINK13　to　downregulate　the　PI3K/Akt　signaling　pathway.　Our　study　helps　elucidate　the　underlying　mechanism　of　SPINK13　in　anti-hepatocellular　carcinoma　and　lays　a　theoretical　foundation　for　the　development　of　novel　therapeutic　serine　protease　inhibitors.　©　The　Author(s)　2024.