AbstractA Cannabinoid Receptor 1 (CB₁) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB₁ affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB₁ trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB₁ enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB₁ modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.