Nonalcoholic fatty liver disease is considered a disorder of the endoplasmic reticulum (ER) and mitochondria. Recent studies have shown that the ER and mitochondrial membranes overlap by 15-20%, a region referred to as the 'mitochondria-associated ER membrane' (MAM). Some proteins, including sarco/ER calcium ATPase (SERCA), are located in the MAM and have an important role in Ca2+ signaling and homeostasis between the ER and the mitochondria. Our previous study showed that hepatic stimulator substance (HSS) inhibits the ER stress induced by reactive oxygen species, thus reducing mitochondrial damage. However, the mechanism underlying the protective effect of HSS on the ER and ER-mitochondrial interaction remains unclear. In this study, we confirmed that the exogenous expression of HSS protected the liver from steatosis in mice with nonalcoholic steatohepatitis. More importantly, the protection provided by HSS allowed SERCA in the MAM compartment to function well, preventing the extensive influx of cytosolic free Ca2+ to the mitochondria, thus preserving the mitochondrial functions from calcium overload and relieving palmitic-acid-induced hepatocyte steatosis. Our results suggest that the protective effect of HSS on SERCA expression is associated with the maintenance of calcium homeostasis within the MAM, thus ameliorating the disordered Ca2+ communication between the ER and mitochondria.