Little is known about the role of microRNA-29a-3p (miR-29a-3p) in inflammation-related pyroptosis, especially in drug-induced acute liver failure (DIALF). This study aimed to identify the relationship between miR-29a-3p and inflammation-related pyroptosis in DIALF and confirm its underlying mechanisms.Thioacetamide (TAA)- and acetaminophen (APAP)-induced ALF mouse models were established, and human samples were collected. The expression levels of miR-29a-3p and inflammation and pyroptosis markers were measured by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, or immunochemical staining in miR-29a-3p knock-in transgenic mouse (MIR29A(KI/KI)) DIALF models. In addition, RNA sequencing was conducted to explore the mechanisms.MiR-29a-3p levels were decreased in TAA- and APAP-induced DIALF models. MiR-29a-3p prevented DIALF caused by TAA and APAP. RNA sequencing and further experiments showed that the protective effect of miR-29a-3p on DIALF was mainly achieved through inhibition of inflammation-related pyroptosis, and the inhibition was dependent on activation of the PI3K/AKT pathway. In addition, miR-29a-3p levels were reduced, and pyroptosis was activated in both peripheral blood mononuclear cells and liver tissues of DIALF patients.The study supports the idea that miR-29a-3p inhibits pyroptosis by activating the PI3K/AKT pathway to prevent DIALF. MiR-29a-3p may be a promising therapeutic target for DIALF.© 2023. Huazhong University of Science and Technology.