We found that global heterozygous midnolin knockout attenuated the severity of nonalnonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose. This attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Overall design: In order to investigate the role of midnolin in NAFLD, we generated midnolin (Midn) global knockout (KO) mice. Global homozygous Midn KO mice, however, were largely embryonic lethal, thus, heterozygous (hemizygous) mice were used for this study. To establish the NAFLD heterozygous Midn KO mouse model, two groups of mice heterozygous (het) and wild-type (wt) were fed with a Western-style diet, high in fat, cholesterol, and fructose ad libitum for 25 weeks. During the special diet feeding, mice were monitored regularly and 5 h-fasting blood was also collected for further serum analysis. At terminal stage, mice's liver tissues were collected and processed for biochemical analysis and histology.