This study aims to investigate the potential mechanism of procyanidin B2 (PB2) against lipid droplets accumulation in vitro and in vivo. Mice were induced to develop lipid metabolism disorders by feeding a high-fat diet (HFD) and induced intracellular lipid droplets deposition with free fatty acids. They were intervened by the administration of PB2 at different concentrations. Medium and high doses of PB2 (75 and 100 mg/kg) significantly reduced body weight and liver index and improved serum and liver lipid profile in HFD-induced mice (P < 0.01 or P < 0.001). In vitro results showed that PB2 decreased intracellular triglyceride content (P < 0.001). Oil red O staining showed that PB2 alleviated lipid droplets accumulation and reduced their number and density in vitro and in vivo. Western blot analysis showed that medium and high doses of PB2 down-regulated the expression of perilipin 5 (Plin5) (P < 0.01 or P < 0.001). In addition, the expression levels of the upstream proteins of Plin5, peroxisome proliferator-activated receptor α (PPARα) and phosphatidylinositol 3-kinase (PI3K) phosphorylation, were inhibited. The above results suggest that lipolysis of lipid droplets mediated through the PI3K/PPARα/Plin5 pathway may be one of the important mechanisms by which PB2 attenuates the excessive accumulation of lipid droplets.