The pathogenesis of non-alcoholic steatohepatitis (NASH) is still unclear and methods for prevention of the development of hepatocellular carcinoma (HCC) have not been established. We established an atherogenic and high-fat diet mouse model that develops hepatic steatosis, inflammation, fibrosis, and liver tumors at a high frequency. Using two NASH-HCC mouse models, we showed that peretinoin, an acyclic retinoid, significantly improved liver histology and reduced the incidence of liver tumors. We performed a microarray method that can comprehensively evaluate RNA expression using the peretinoin-treated mouse liver tissue. Through MetaCore software, which enables bioinformatic analysis using the expression data obtained by the microarray method, we discovered the possibility of inducing the activity of autophagy, a new physiological function of peretinoin. This was characterized by increased colocalized expression of microtubule-associated protein light chain 3B-II and lysosome-associated membrane protein 2, and increased autophagosome formation and autophagic flux. Among representative autophagy pathways, the autophagy related (Atg) 5-Atg12-Atg16L1 pathway was impaired; especially, Atg16L1 was repressed at both the mRNA and protein level. Decreased Atg16L1 mRNA expression was also found in the liver of patients with NASH according to disease progression. Thus, peretinoin prevents the progression of NASH and the development of HCC through activating the autophagy pathway by increased Atg16L1 expression, which is an essential regulator of autophagy and anti-inflammatory proteins. (Oncotarget, 2017, Vol. 8, (No. 25), pp: 39978-39993).