Cell migration is key to tumor metastasis, the most challenging clinical problem in cancer treatment. Here we are investigating the role of DENND10/FAM45A, a recently described endosomal protein, in cancer cell migration via modulating the release of extracellular vesicles (EVs). Bioinformatics data mining showed that high DENND10 expression is significantly associated with poor prognosis in multiple cancer types. DENND10 knockout (DENND10-KO) resulted in defective cell spreading and migration, with impaired formation of stress fibers and focal adhesions. EV secretion was reduced in DENND10-KO cells. Importantly, wild-type conditioned medium or EVs restored the migratory ability and cytoskeletal organization of DENND10-KO cells. Quantitative mass spectrometry revealed that DENND10 depletion led to distinct EV compositional profiles with remodeled constituents of adhesion molecules and extracellular matrix. Together, our study unveiled DENND10 as an intrinsic coordinator between EV release and cell migration, which could be exploited for designing new anti-cancer strategies.