Extracellular signals have to be precisely interpreted intracellularly and translated into diverse cellular behaviors often mediated by cytoskeletal changes. Semaphorins are one of the largest families of guidance cues and play a critical role in many systems. However, how different cell types translate extracellular semaphorin binding into intracellular signaling remains unclear. Here we developed and performed a novel image-based genome-wide functional RNAi screen for downstream signaling molecules that convert the interaction between Semaphorin 3E (Sema3E) and PlexinD1 into cellular behaviors. One of the genes identified in this screen is a RhoGAP protein, SH3-domain binding protein 1 (SH3BP1). We demonstrate that SH3BP1 mediates Sema3E-induced cell collapse through interaction with PlexinD1 and regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1) activity. The identification and characterization of SH3BP1 as a novel downstream effector of Sema3E-PlexinD1 provides an explanation for how extracellular signals are translated into cytoskeletal changes and unique cell behavior, but also lays the foundation for characterizing other genes identified from our screen to obtain a more complete picture of plexin signaling.