Alternative splicing provides a means to increase the complexity of gene function in numerous biological processes, including nervous system wiring. Navigating axons switch responses from attraction to repulsion at intermediate targets, allowing them to grow to each intermediate target and then to move on. The mechanisms underlying this switch remain poorly characterized. We previously showed that the Slit receptor Robo3 is required for spinal commissural axons to enter and cross the midline intermediate target. We report here the existence of two functionally antagonistic isoforms of Robo3 with distinct carboxy termini arising from alternative splicing. Robo3.1 is deployed on the precrossing and crossing portions of commissural axons and allows midline crossing by silencing Slit repulsion. Robo3.2 becomes expressed on the postcrossing portion and blocks midline recrossing, favoring Slit repulsion. The tight spatial regulation of opponent splice variants helps ensure high-fidelity transition of axonal responses from attraction to repulsion at the midline.