Ubiquitin-specific protease 14 (USP14) is one of the major proteasome-associated deubiquitinating enzymes critical for proteome homeostasis. However, substrates of USP14 remain largely unknown, hindering the understanding of its functional roles. Here we conduct a comprehensive proteome, ubiquitinome and interactome analysis for USP14 substrate screening. Bioinformatics analysis reveals broad new potential roles of USP14, especially in lipid and carbohydrate metabolism. Among the potential substrates identified, we show that fatty acid synthase (FASN), a key enzyme involved in hepatic lipogenesis, is a bona fide substrate of USP14. USP14 directly interacts with and increases FASN stability. As a result, overexpression of USP14 promotes liver triglyceride accumulation in C57BL/6 mice, whereas genetic ablation or pharmacological inhibition of USP14 ameliorates hepatosteatosis, hyperglycemia and insulin resistance in obese mice. In conclusion, our findings reveal for the first time an indispensable role of USP14 in hepatosteatosis through FASN stabilization.