Non-alcoholic fatty liver disease (NAFLD), characterized by the accumulation of fat within hepatocytes, has become the most common chronic liver disease in developed countries. NAFLD is associated with insulin resistance, obesity, and cardiovascular disease. In this work, the role of hydroxysteroid 17-beta dehydrogenase (HSD17B) type-12 and -13 in the development of fatty liver disease was investigated using knockout (KO) mouse models. Previous results have shown that HSD17B13 is mostly expressed in the liver on the surface of lipid droplets. In humans, certain HSD17B13 variants have been found to protect against NAFLD. HSD17B12, on the other hand, is highly expressed in metabolically active tissues, including the liver, and previous studies suggest that the enzyme is involved in lipid metabolism. However, the exact function of either enzyme in liver cells is still unclear. HSD17B13 deficiency led to fat accumulation in hepatocytes and hepatic inflammation in 9-month-old male mice. Inhibition of HSD17B12 enzyme activity in the whole body of adult mice led to the development of fatty liver, but also to very rapid weight loss. The observed weight loss in mice was associated with reduced food intake. Similar but slower changes were also observed in hepatocyte-specific HSD17B12cKO mice. The results indicate that the functions of HSD17B13 and HSD17B12 enzymes are related to cellular mechanisms that regulate the formation and growth of hepatic lipid droplets. The molecular mechanisms of both enzymes remain to be elucidated, but our results and those of others suggest that HSD17B13 and HSD17B12 are promising targets for the development of drug therapies for fatty liver disease.