Fatty liver is the most common type of liver disease, affecting nearly one third of the US population and more than half a billion people worldwide. Abnormalities in ER calcium handling and mitochondrial function each have been implicated in abnormal lipid droplet formation. Here we show that the type 1 isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R1) specifically links ER calcium release to mitochondrial calcium signaling and lipid droplet formation in hepatocytes. Moreover, liver-specific InsP3R1 knockout mice have impaired mitochondrial calcium signaling, decreased hepatic triglycerides, reduced lipid droplet formation and are resistant to development of fatty liver. Patients with non-alcoholic steatohepatitis, the most malignant form of fatty liver, have increased hepatic expression of InsP3R1 and the extent of ER-mitochondrial co-localization correlates with the degree of steatosis in human liver biopsies.ConclusionInsP3R1 plays a central role in lipid droplet formation in hepatocytes and the data suggest that it is involved in the development of human fatty liver disease.