Cannabinoid receptor 1 (CB₁) is an abundant G protein-coupled receptor, involved in a number of physiological processes. This receptor is localized at the plasma membrane, as well as in intracellular vesicles. The trafficking events leading to this intracellular localization remain controversial. In this study, we examine the differential trafficking of CB₁ receptors and its implication on signaling. We find that the transfected tagged receptors are predominantly at the plasma membrane, whereas endogenous receptors exhibit an intracellular localization. We also find that intracellular endogenous CB₁ receptors do not have an endocytic origin. Instead, these receptors associate with the adaptor protein AP-3 and traffic to the lysosomes. siRNA-mediated AP-3 knockdown leads to enhanced cell surface localization of CB₁ receptors. Finally, we show that CB₁ receptors in the late endosomal/lysosomal compartment are associated with heterotrimeric G proteins and mediate signal transduction. These results suggest that intracellular CB₁ receptors are functional and that their spatial segregation is likely to significantly affect receptor function.—Rozenfeld, R., Devi, L. A. Regulation of CB₁ cannabinoid receptor trafficking by the adaptor protein AP-3.