S14 has been identified as a potent stimulator of de novo hepatic lipogenesis (DNL) in rodents. However, it is unclear how S14 is regulated in humans with non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship between serum S14 and liver steatosis in humans with NAFLD. A total of 614 participants were recruited from community. Liver steatosis were evaluated according to the Ultrasonographic Fatty Liver Indicator (US-FLI), which is a semi-quantitative liver ultrasound score. Anthropometric and biochemical indices were collected for further analysis. The risk of liver steatosis severity was estimated by a cumulative logistic regression model. NAFLD was found in 52.2% of the participants. The subjects with NAFLD showed higher levels of waist circumference, body mass index, insulin resistance, aspartate aminotransferase, dyslipidemia, visceral fat, serum S14 and risk of metabolic syndrome (MetS) than those of controls. Compared with the first tertile of serum S14, the odds ratios for the risk of more severe liver steatosis were 1.22 (95% confidence interval [CI]: 0.78–1.92) for those of the second tertile and 2.08 (95% CI: 1.28–3.39) for the third tertile (P for trend < 0.05) after adjusting for confounding factors. Higher serum S14 level was not only found in NAFLD subjects but also was positively correlated with the severity of liver steatosis. S14 may play an important role in the mechanism of DNL for NAFLD in humans.