The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB₁ cannabinoid receptors (CB₁Rs) are enriched in the axonal growth cones of γ-aminobutyric acid–containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB₁R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB₁Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.