Long-term synaptic plasticity is critical for adaptive function of the brain, but presynaptic mechanisms of functional plasticity remain poorly understood. Here, we show that changes in synaptic efficacy induced by activation of the cannabinoid type-1 receptor (CB 1 R), one of the most widespread G-protein coupled receptors in the brain, requires contractility of the neuronal actomyosin cytoskeleton. Specifically, using a synaptophysin-pHluorin probe (sypH2), we show that inhibitors of non-muscle myosin II (NMII) ATPase as well as one of its upstream effectors Rho-associated kinase (ROCK) prevent the reduction of synaptic vesicle release induced by CB 1 R activation. Using 3D STORM super-resolution microscopy, we find that activation of CB 1 R induces a redistribution of synaptic vesicles within presynaptic boutons in an actomyosin dependent manner, leading to vesicle clustering within the bouton and depletion of synaptic vesicles from the active zone. We further show, using sypH2, that inhibitors of NMII and ROCK specifically restore the release of the readily releasable pool of synaptic vesicles from the inhibition induced by CB 1 R activation. Finally, using slice electrophysiology, we find that activation of both NMII and ROCK is necessary for the long-term, but not the short-term, form of CB 1 R induced synaptic plasticity at excitatory cortico-striatal synapses. We thus propose a novel mechanism underlying CB 1 R-induced plasticity, whereby CB 1 R activation leads to a contraction of the actomyosin cytoskeleton inducing a reorganization of the functional presynaptic vesicle pool, preventing vesicle release and inducing long-term depression.