Betatrophin/angiopoietin-like protein 8 (ANGPTL8) is a liver-secreted protein recently identified as a potent stimulator of beta cell proliferation in mice. However, it is unclear how betatrophin is regulated in humans with non-alcoholic fatty liver disease (NAFLD). We investigated the role of betatrophin in mice and in humans with and without NAFLD. Serum betatrophin levels were examined by ELISA in 164 subjects, including 96 patients with NAFLD. Levels were significantly elevated in subjects with NAFLD compared with controls (1.301 ± 0.617 vs. 0.900 ± 0.574 μg/L, P < 0.001), even after stratification by diabetic or obesity status. Circulating betatrophin positively correlated with obesity or glycemic indices, liver enzyme profiles, and NAFLD status, and was confirmed by multivariate regression analyses (β = 0.195, P = 0.040). However, when including insulin resistance index in the model, the significant association between betatrophin level and NAFLD was diminished due to a mediation effect of insulin resistance on this relationship. Palmitate or tunicamycin increased betatrophin expression in HepG2 cells, while a chemical chaperone blocked its induction. Hepatic expression of betatrophin was elevated in mice with NAFLD including db/db or ob/ob mice and mice with a high-fat or methionine-choline deficient diet. In conclusion, circulating betatrophin was increased in mice and humans with NAFLD and its expression was induced by endoplasmic reticulum stress in hepatocytes (Clinical trial no. NCT02285218).