Hepatic lipid droplets (LDs) are implicated in ectopic lipid accumulation. The core of LDs, triacylglycerol (TAG), is synthesized from the esterification of fatty acids to a glycerol-3-phosphate (G-3-P) backbone. Albumin transports plasma free fatty acids, and previously albumin knockout (Alb−/−) mice were shown to exhibit lower hepatic TAG levels than wildtype (WT). Exercise is a beneficial strategy to alter hepatic metabolism, but its impacts on reducing hepatic lipids are far from satisfactory. The aim of this study was to investigate the combined effect of albumin deficiency and acute exercise on hepatic LDs. Eight-week-old male Alb−/−and WT mice were divided into sedentary and exercise groups. Exercised mice performed a 30-min high-intensity exercise bout. Results showed that sedentary Alb−/−mice had smaller hepatic LDs (P< 0.0001), associated with mitochondria, while WT mice exhibited larger LDs, surrounded by glycogen granules. Following acute exercise, hepatic LDs in Alb−/−mice reduced by 40% in size, while in WT increased by 14% (P< 0.0001). The maintenance of WT hepatic LDs was associated with elevated G-3-P level (P< 0.05), potentially derived from glycogen (R = -0.32, %change in glycogen versus LD content,P< 0.05). The reduction in Alb−/−mice LDs after exercise was possibly due to their low glycogen level. In conclusion, Alb−/−mice exhibited an enhanced capacity for reducing hepatic LD size and content in response to exercise. These findings suggest that modulating albumin’s functions combined with exercise could be a potential strategy to reduce ectopic lipid deposition in the liver.