Fatty acid translocase cluster of differentiation (CD36) is a multifunctional membrane protein that facilitates the uptake of long-chain fatty acids. Lipophagy is autophagic degradation of lipid droplets. Accumulating evidence suggests that CD36 is involved in the regulation of intracellular signal transduction that modulates fatty acid storage or usage. However, little is known about the relationship between CD36 and lipophagy. In this study, we found that increased CD36 expression was coupled with decreased autophagy in the livers of mice treated with a high-fat diet. Overexpressing CD36 in HepG2 and Huh7 cells inhibited autophagy, while knocking down CD36 expression induced autophagy due to the increased autophagosome formation in autophagic flux. Meanwhile, knockout of CD36 in mice increased autophagy, while the reconstruction of CD36 expression in CD36-knockout mice reduced autophagy. CD36 knockdown in HepG2 cells increased lipophagy and β-oxidation, which contributed to improving lipid accumulation. In addition, CD36 expression regulated autophagy through the AMPK pathway, with phosphorylation of ULK1/Beclin1 also involved in the process. These findings suggest that CD36 is a negative regulator of autophagy, and the induction of lipophagy by ameliorating CD36 expression can be a potential therapeutic strategy for the treatment of fatty liver diseases through attenuating lipid overaccumulation.