LPS has been shown to induce hepatocyte autophagy, but little is known about how LPS is able to do this during acute toxic liver injury. Our aim was to determine the existence of any selective Ca 2+ signaling coupling to hepatocyte autophagy in response to LPS. LPS increased the autophagic process in hepatocytes, and CD38 knockdown prevented this response. Ned19, a specific inhibitor for nicotinic acid adenine dinucleotide phosphate (NAADP), prevented LPS-mediated Ca 2+ signaling and autophagosome formation in hepatocytes. CD38 overexpression protected the liver from LPS/d-galactosamine (GalN)-induced injury, and NAADP administration promoted autophagosome formation and protected hepatocytes from injury induced by LPS/GalN. Autophagy was promoted by the up-regulation of autophagy-related gene expression via NAADP-mediated Ca 2+ signaling in response to LPS. However, CD38-knockout mice displayed down-regulation in hepatocyte gene expression. Ned19 also inhibited the NAADP-stimulated induction of gene expression by inhibiting the LPS-induced nuclear translocation of transcription factor EB (TFEB). Hepatocyte autophagy protects against LPS-induced liver injury via the CD38/NAADP/Ca 2+/TFEB pathway. The role of NAADP-mediated Ca 2+ signaling in the autophagic process will help elucidate the complexities of autophagy regulation, which is essential toward the discovery of new therapeutic tools against acute liver injury.—Rah, S.-Y., Lee, Y.-H., Kim, U.-H. NAADP-mediated Ca 2+ signaling promotes autophagy and protects against LPS-induced liver injury.