Non-alcoholic fatty liver disease (NAFLD) is becoming a severe liver disorder worldwide. Autophagy plays a critical role in liver steatosis. However, the role of autophagy in NAFLD remains exclusive and under debate. In this study, we investigated the role of S100 calcium binding protein A11 (S100A11) in the pathogenesis of hepatic steatosis. We performed liver proteomics in a well-established tree shrew model of NAFLD. The expression of S100A11 in different models of NAFLD was detected by Western blotting and/or qPCR. Liver S100A11 overexpression mice were generated by injecting a recombinant adenovirus gene transfer vector through the tail vein and then induced by a high-fat and high-cholesterol (HFHC) diet. Cell lines with S100a11 stable overexpression were established with a recombinant lentiviral vector. The lipid content was measured with either Bodipy staining, Oil Red O staining, gas chromatography, or a triglyceride kit. The autophagy and lipogenesis were detected in vitro and in vivo by Western blotting and qPCR. The functions of SIRT1, HDAC6, and FOXO1 were inhibited by specific inhibitors. The interactions between related proteins were analyzed by a Co-IP assay and immunofluorescence analysis. The expression of S100A11 significantly upregulated in a time-dependent manner in the tree shrew model of NAFLD. S100A11 expression was consistently induced in oleic acid treated liver cells as well as the livers of mice fed a high-fat diet and NAFLD patients. Both in vitro and in vivo overexpression of S100A11 could induce hepatic lipid accumulation. Mechanistically, overexpression of S100A11 activated an autophagy and lipogenesis process through upregulation and acetylation of the transcriptional factor FOXO1, consequently promoting lipogenesis and lipid accumulation in vitro and in vivo. Inhibition of HDAC6, a deacetylase of FOXO1, exhibited similar phenotypes to S100A11 overexpression in Hepa 1-6 cells. S100A11 interacted with HDAC6 to inhibit its activity, leading to the release and activation of FOXO1. Under S100A11 overexpression, the inhibition of FOXO1 and autophagy could alleviate the activated autophagy as well as upregulated lipogenic genes. Both FOXO1 and autophagy inhibition and Dgat2 deletion could significantly reduce liver cell lipid accumulation. A high-fat diet promotes liver S100A11 expression, which may interact with HDAC6 to block its binding to FOXO1, releasing or increasing the acetylation of FOXO1, thus activating autophagy and lipogenesis, and accelerating lipid accumulation and liver steatosis. These findings indicate a completely novel S100A11-HDAC6-FOXO1 axis in the regulation of autophagy and liver steatosis, providing potential possibilities for the treatment of NAFLD. Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.