Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology. SIGNIFICANCE: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.See related commentary by McAndrews and Kalluri, p. 2099.