Lipid droplets are intracellular organelles that provide a depot for triglyceride storage in almost all cells and are a central point for the control of cellular energy homeostasis. Defective lipid droplet metabolism is a common occurrence in the pathogenesis of obesity, non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D). Perilipin (PLIN) 5 regulates lipid metabolism by co-ordinating protein-protein interactions at the surface of lipid droplets. We rationalised that other proteins located at the lipid droplet might interact with PLIN5 to regulate lipid metabolism. To test this, we affinity purified PLIN5 tagged with 3XHA from cell homogenates and identified potential interacting proteins using mass spectrometry analysis. Steroid hormone enzyme 17-β-hydroxysteroid dehydrogenase type 11 (HSD17β11) was the most abundant interacting partner of PLIN5 and was subsequently confirmed as a bone fide lipid droplet localised protein using confocal microscopy. We next employed coimmunoprecipitation and live cell imaging approaches to show that HSD17β11 interacts with ATGL and PLIN5, which are important regulators of lipolysis. CRISPR/Cas9 gene editing of HSD17β11 in cells resulted in the accumulation of enlarged lipid droplets, which was mediated by reduced lipolysis and fatty acid b-oxidation. Re-expression of HSD17β11 in HSD17β11-null cells reversed these effects on lipid metabolism. Gene expression analysis in human liver biopsies revealed downregulation of HSD17β11 mRNA expression in patients with non-alcoholic steatohepatitis (NASH) and T2D compared with weight and age-matched individuals, and HSD17β11 expression was negatively associated with hepatic steatosis. All together, these results demonstrate an important role of HSD17β11 in regulating cellular lipid metabolism and indicate a potential role HSD17β11 in the pathogenesis of fatty liver disease. We have produced HSD17β11 null mice using CRISPR/Cas9 gene editing and ongoing studies aim to delineate HSD17β11’s metabolic functions in vivo.