Here, we investigated the importance of hormone-sensitive lipase (HSL) as a retinyl ester hydrolase (REH). REH activity was measured in vitro using recombinant HSL and retinyl palmitate. The expression of retinoic acid (RA)-regulated genes and retinoid metabolites were measured in high-fat diet fed HSL-null mice using real-time quantitative PCR and triple-stage liquid chromatography/tandem mass spectrometry, respectively. Age- and gender-matched wild-type littermates were used as controls. The REH activity of rat HSL was found to be higher than that against the hitherto best known HSL substrate, i.e., diacylglycerols. REH activity in white adipose tissue (WAT) of HSL-null mice was completely blunted and accompanied by increased levels of retinyl esters and decreased levels of retinol, retinaldehyde and all-trans RA. Accordingly, genes known to be positively regulated by RA were down-regulated in HSL-null mice, including pRb and RIP140, key factors promoting differentiation into the white over the brown adipocyte lineage. Dietary RA supplementation partly restored WAT mass and the expression of RA-regulated genes in WAT of HSL-null mice. These findings demonstrate the importance of HSL as an REH of adipose tissue and suggest that HSL via this action provides RA and other retinoids for signaling events that are crucial for adipocyte differentiation and lineage commitment.—Ström, K., Gundersen, T. E., Hansson, O., Lucas, S., Fernandez, C., Blomhoff, R., Holm, C. Hormone-sensitive lipase (HSL) is also a retinyl ester hydrolase: evidence from mice lacking HSL.