Metabolic dysfunction-associated fatty liver disease (MAFLD) is severe and widespread. While obesity and Western diets are known risk factors for MAFLD, identifying the environmental and genetic modifiers causing its heterogeneous onset and progression is critical to elucidating its mechanisms and developing intervention strategies. Several lines of evidence suggest reciprocal relationships between MAFLD and the dysregulation of copper, a cofactor of approximately a dozen enzymes, such as cytochrome oxidase and cytosolic superoxide dismutase. This project aimed to gain better insight into the roles of copper in lipid metabolism and metabolic diseases. We determined excess fat-induced changes in copper homeostasis and the impacts of copper limitation on lipid metabolism in the mouse liver and cultured hepatocytes. Our data present a fatty acid-dependent expression of copper-requiring enzymes and molecular factors involved in copper homeostasis. Furthermore, copper limitation in hepatocytes rapidly developed severe lipid toxicity and whole-body lipid metabolism disorders. These results indicate the critical roles of copper-dependent cellular processes in lipid metabolism and provide a new conceptual framework to define previously under-appreciated contributions of copper and other micronutrients in preventing metabolic diseases.