During central nervous system development in both vertebrates and invertebrates, commissural neurons must project their axons across the midline to form synaptic connections on the opposite side of the body. Midline crossing allows for coordination between the body’s left and right sides, and aberrant crossing is implicated in various neurodevelopmental disorders affecting motor and cognitive function. During midline crossing, axons follow a stereotyped projection pattern, directed by conserved axon guidance ligands secreted by midline glia which interact with receptors on the growth cone. Initially, axons are attracted to the midline but become repelled upon reaching it, to allow for exit from the midline and prevent recrossing. As midline glia secrete both attractive and repulsive ligands simultaneously, surface expression of axon guidance receptors must be tightly regulated for axons to display attractive or repulsive behavior at the appropriate time. In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. Here, we demonstrate that conserved PY motifs in the Comm cytoplasmic domain are required to allow ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1-positive late endosomes and prevents midline crossing in vivo. In addition, we conclusively demonstrate a role for the Nedd4 ubiquitin ligase in midline crossing. Genetic analysis shows that nerve cords of nedd4 mutant Drosophila embryos have midline crossing defects, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a ternary complex with Comm and Robo1 in a PY motif-dependent manner. Finally, we present genetic evidence that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo1 ubiquitination by Nedd4, ultimately leading to its degradation. As Nedd4 was recently implicated in vertebrate Robo1 downregulation, these findings also point to a conserved mechanism guiding axon behavior across the midline.