Objective To investigate the effect of protein phosphatase 5 (PP5) on lipid metabolism in the PP5 knockout (KO) mice. Methods Male PP5 KO and wild type (WT) mice at the age of 6 weeks were used in this study. In order to study the effect of high fat diet (HFD) feeding, the body weight was measured. The liver histology was examined by HE and oil red O staining. To further verify PP5 functions in the adipogenesis, in vitro experiment was carried out using mouse embryonic fibroblasts (MEF). Western blotting and real-time PCR were performed to quantified the expression of lipid metabolism-related genes in the liver tissues.Results Compared with the WT mice, the body weight gain was slower in the KO mice. The size of the lipid droplets was smaller and the quantity was less in the KO mouse liver tissue. In vitro study revealed that the KO mouse MEF cells showed less differentiated adipocytes with smaller lipid droplets than the WT MEF cells. This observation was further confirmed by detecting the expression of adipogenesis-related genes in the HFD liver. The markers of adipocyte differentiation, such as CD36, AP2, PPARγ2, and Glut4, were significantly decreased, while energy expenditure-related markers, such as phosphorylation of GR and expression of UCP1, were significantly increased. Conclusions Protein phosphatase 5 may play a regulatory role in the mouse lipid metabolism through regulating the de-phosphorylation of p-GR and enhancing the expression of UCP1.