Hydroxysteroid (17β) dehydrogenases (HSD17Bs) form an enzyme family characterized by their ability to catalyze reactions in steroid and lipid metabolism. In the present study, we characterized the phenotype of HSD17B13‐knockout (HSD17B13KO) mice deficient in Hsd17b13. In these studies, hepatic steatosis was detected in HSD17B13KO male mice, indicated by histologic analysis and by the increased triglyceride concentration in the liver, whereas reproductive performance and serum steroid concentrations were normal in HSD17B13KO mice. In line with these changes, the expression of key proteins in fatty acid synthesis, such as FAS, acetyl‐CoA carboxylase 1, and SCD1, was increased in the HSD17B13KO liver. Furthermore, the knockout liver showed an increase in 2 acylcarnitines, suggesting impaired mitochondrial β‐oxidation in the presence of unaltered malonyl CoA and AMPK expression. The glucose tolerance did not differ between wild‐type and HSD17B13KO mice in the presence of lower levels of glucose 6‐phosphatase in HSD17B13KO liver compared with wild‐type liver. Furthermore, microgranulomas and increased portal inflammation together with up‐regulation of immune response genes were observed in HSD17B13KO mice. Our data indicate that disruption of Hsd17b13 impairs hepatic‐lipid metabolism in mice, resulting in liver steatosis and inflammation, but the enzyme does not play a major role in the regulation of reproductive functions.—Adam M., Heikelä H., Sobolewski, C., Portius, D., Mäki‐Jouppila J., Mehmood, A., Adhikari, P., Esposito, I., Elo, L. L., Zhang, F.‐P., Ruohonen, S. T., Strauss, L., Foti, M., Poutanen M. Hydroxysteroid (17β) dehydrogenase 13 deficiency triggers hepatic steatosis and inflammation in mice. FASEB J. 32, 3434–3447 (2018). www.fasebj.org