HSD17B13 (17-beta hydroxysteroid dehydrogenase 13) is genetically associated with human non-alcoholic fatty liver disease (NAFLD). Inactivating mutations in HSD17B13 protect humans from NAFLD- and alcohol-associated liver injury, fibrosis, cirrhosis and hepatocellular carcinoma, leading to clinical trials of anti-HSD17B13 therapeutic agents in humans. We aimed to study the in vivo function of HSD17B13 using a mouse model. Single-cell RNAseq and qPCR data revealed that hepatocytes are the main HSD17B13-expressing cells in mice and humans. We compared Hsd17b13 whole-body knockout (KO) mice and wild-type littermate controls (WT) fed regular chow (RC), high fat diet (HFD), Western diet (WD), or the NIAAA model of alcohol exposure. HFD and WD induced significant weight gain, hepatic steatosis and inflammation. However, there was no difference between genotypes with regards to body weight, liver weight, hepatic triglycerides (TG), histological inflammatory scores, expression of inflammatory- and fibrosis-related genes and hepatic retinoid levels. Compared to WT, KO mice on HFD had hepatic enrichment of most cholesterol esters, monoglycerides, and certain sphingolipids species. Extended feeding with WD for 10 months led to extensive liver injury, fibrosis and hepatocellular carcinoma, with no difference between genotypes. Under alcohol exposure, KO and WT mice showed similar hepatic TG and liver enzyme levels. Interestingly, chow-fed KO showed significantly higher body and liver weight compared to WT, while KO mice on obesogenic diets had a shift towards larger lipid droplets. In conclusion, extensive evaluation of Hsd17b13 deficiency in mice under several fatty liver-inducing dietary conditions didn't reproduce the protective role of HSD17B13 loss-of-function mutants in human NAFLD. Moreover, mouse Hsd17b13 deficiency induces weight gain under RC. It is crucial to understand inter-species differences prior to leveraging HSD17B13 therapies.