<p>Human umbilical cord mesenchymal stromal cells (hucMSCs) have emerged as a promising therapeutic option for acute liver failure (ALF). However, the detailed mechanism by which hucMSCs modulate hepatocyte pyroptosis in ALF remains unclear. In this study, we induced ALF in mice using acetaminophen (APAP). Mice were intravenously injected with 1 × 10⁶ hucMSCs/ hucMSCs-Exo via the tail vein 6 h after APAP administration. Liver pathological changes were assessed by hematoxylin and eosin (H&amp;E) staining. Subsequently, an in vitro model of liver cell failure and pyroptosis model was established using LO2 cells treated with lipopolysaccharide (LPS) and adenosine 5’-triphosphate (ATP). The levels of cell pyroptosis marker caspase-1 were detected by flow cytometry analysis, RT-qPCR assay, and western blot assay. The study employed a comprehensive approach, including flow cytometry analysis, RT-qPCR assay, miRNA sequencing, and luciferase reporter gene experiments. hucMSCs and hucMSCs- exosomes (MSCs-Exo) inhibited cell inflammation to improve ALF in vivo model of ALF and inhibited hepatocyte pyroptosis in LO2 cells induced by ATP and LPS. MiR-423-5p emerged as a potential mediator of the anti-pyroptotic effects of hucMSCs-Exo, with ZBP1 identified as one of its downstream targets. Subsequent validation confirmed that miR-423-5p targets ZBP1 to regulate pyroptosis. These findings highlight the role of hucMSCs-derived exosomal miR-423-5p in inhibiting hepatocyte pyroptosis in LO2 cells induced by ATP and LPS. miR-423-5p serves as a crucial mediator in this process, targeting ZBP1, a protein significantly involved in the pyroptotic pathway. Our findings may provide basics for further research on mechanism of hucMSCs and present a promising cell-free strategy treating ALF.</p>