Members of the Slit family of secreted ligands interact with Roundabout (Robo) receptors to provide guidance cues for many cell types. For example, Slit/Robo signaling elicits repulsion of axons during neural development, whereas in endothelial cells this pathway inhibits or promotes angiogenesis depending on the cellular context. Here, we show that miR-218 is intronically encoded in slit2 and slit3 and that it suppresses Robo1 and Robo2 expression. Our data indicate that miR-218 and multiple Slit/Robo signaling components are required for heart tube formation in zebrafish and that this network modulates the previously unappreciated function of Vegf signaling in this process. These findings suggest a new paradigm for microRNA-based control of ligand-receptor interactions and provide evidence for a novel signaling pathway regulating vertebrate heart tube assembly.