During Drosophila metamorphosis, the single-cell layer of fat body tissues gradually dissociates into individual cells. Via a fat body-specific RNAi screen in this study, we found that two matrix metalloproteinases (MMPs), Mmp1 and Mmp2, are both required for fat body cell dissociation. As revealed through a series of cellular, biochemical, molecular, and genetic experiments, Mmp1 preferentially cleaves DE-cadherin-mediated cell-cell junctions, while Mmp2 preferentially degrades basement membrane (BM) components and thus destroy cell-BM junctions, resulting in the complete dissociation of the entire fat body tissues into individual cells. Moreover, several genetic interaction experiments demonstrated that the roles of Mmp1 and Mmp2 in this developmental process are cooperative. In conclusion, Mmp1 and Mmp2 induce fat body cell dissociation during Drosophila metamorphosis in a cooperative yet distinct manner, a finding that sheds light on the general mechanisms by which MMPs regulate tissue remodeling in animals.