Slit regulates migration of not only neurons, but also nonneuronal cells, such as leukocytes and cancer cells. Slit effect on cancer cell migration has not been well-characterized. In this study, we used several different assays to examine Slit effect on breast cancer cell migration in vitro. We show that ubiquitin-specific protease 33 (USP33)/VDU1, originally identified as a von Hippel-Lindau tumor suppressor (VHL) protein-interacting deubiquitinating enzyme, binds to the Robo1 receptor, and that USP33 is required for Slit responsiveness in breast cancer cells. Slit induces redistribution of Robo1 from intracellular compartments to the plasma membrane in a USP33-dependent manner. Slit impairs directional migration of breast cancer cells without affecting their migration speed. This inhibitory effect is Robo-mediated and USP33-dependent. These data uncover a previously unknown function of USP33 and reveal a new player in Slit-Robo signaling in cancer cell migration.