Hydroxysteroid 17β-dehydrogenase 13 (HSD17B13) loss-of-function gene variants are associated with a decreased risk of metabolic dysfunction-associated steatotic liver disease (MASLD). Our RNA-seq analysis of steatotic liver from obese mice ± fenretinide treatment identified major beneficial effects of fenretinide on expression of hepatic genes including Hsd17b13. We sought to determine the relationship between Hsd17b13 expression and MASLD and to validate it as a therapeutic target by liver-specific knockdown. Hsd17b13 expression, which is unique to hepatocytes and associated with the lipid droplet, was elevated in multiple models of MASLD and normalised with the prevention of obesity and steatotic liver. Direct, liver-specific, shRNA-mediated knockdown of Hsd17b13 (shHsd17b13) in high-fat diet (HFD)-obese mice (n = 8–9 male C57BL/6J per group), markedly improved hepatic steatosis with no effect on body weight, adiposity or glycaemia. shHsd17b13 decreased elevated serum alanine aminotransferase (ALT), serum fibroblast growth factor 21 (FGF21) levels, and markers of liver fibrosis, for example, expression of Timp2. shHsd17b13 knockdown in HFD-obese mice and Hsd17b13 overexpression in cells reciprocally regulated expression of lipid metabolism genes, for example, Cd36. Global lipidomic analysis of liver tissue revealed a major decrease in diacylglycerols (e.g. DAG 34:3) with shHsd17b13 expression and an increase in phosphatidylcholines containing polyunsaturated fatty acids (PUFA) for example, phosphatidylcholine (PC) 34:3 and PC 42:10. Expression of key genes involved in phospholipid and PUFA metabolism, for example, Cept1, was also reciprocally regulated suggesting a potential mechanism of Hsd17b13 biological function and role in MASLD. In conclusion, Hsd17b13 knockdown in HFD-obese adult mice was able to alleviate MASLD via regulation of fatty acid and phospholipid metabolism, thereby confirming HSD17B13 as a genuine therapeutic target for MASLD and the development of liver fibrosis.