Hormone-sensitive lipase is one of the rate-determining enzymes in the hydrolysis of triglyceride, playing a crucial role in lipid metabolism. However, the role of HSL-mediated lipolysis in systemic nutrient homeostasis has not been intensively understood. Therefore, we used CRISPR/Cas9 technique and Hsl inhibitor (HSL-IN-1) to establish hsla-deficient (hsla-/-) and Hsl-inhibited zebrafish models, respectively. As a result, the hsla-/- zebrafish showed retarded growth and reduced oxygen consumption rate, accompanied with higher mRNA expression of the genes related to inflammation and apoptosis in liver and muscle. Furthermore, hsla-/- and HSL-IN-1 treated zebrafish both exhibited severe fat deposition, whereas their expressions of the genes related to lipolysis and fatty acid oxidation were markedly reduced. The thin layer chromatography results also showed that the dysfunction of Hsl changed the whole body lipid profile, including increasing the content of TG and decreasing the proportion of PL. In addition, the systemic metabolic pattern was remodeled in hsla-/- and HSL-IN-1 treated zebrafish. The dysfunction of Hsl lowered the glycogen content in liver and muscle, enhanced the utilization of glucose plus the expressions of glucose transporter and glycolysis genes. Besides, the whole-body protein content had significantly decreased in the hsla-/- and HSL-IN-1 treated zebrafish, accompanied with the lower activation of the mTOR pathway and enhanced protein and amino acid catabolism. Taken together, Hsl plays an essential role in energy homeostasis, and its dysfunction would cause the disturbance of lipid catabolism but enhanced breakdown of glycogen and protein for energy compensation.