Background: Desensitization and internalization are key procedures in the regulation of hormone receptors, and beta-arrestins play an important role in these processes. Currently, little is known about the role of beta-arrestins in the regulation of the CB1 cannabinoid receptor (CB1R). Therefore, the interaction between beta-arrestins and CB1R, and its role in receptor internalization were investigated. Materials and methods: The beta-arrestin binding of CB1R was studied by confocal microscopy and bioluminescence resonance energy transfer (BRET) in HeLa and Neuro-2a cells. To follow CB1R internalization, plasma membrane CB1 receptors were selectively stained using Halo-labeling technique. Internalization of the receptors was also monitored by a BRET assay through measuring non-specific BRET between CB1R and ICAM as a plasma membrane marker. Results: We found that upon activation CB1R binds beta-arrestin2 (beta-arr2), but not beta-arrestin1, and this binding is transient. We also found that internalization following stimulation with CB1R agonists could be impaired either by a dominant negative beta-arrestin2 (beta-arr2-V54D) or by siRNA-mediated knock-down of beta-arr2. Similar inhibitory effects on agonist-induced internalization were detected with BRET measurements. In contrast, neither beta-arr2-V54D nor beta-arr2-siRNA had a significant effect on the constitutive (i.e. spontaneous) internalization of CB1R. Conclusions: We conclude that upon activation, CB1R binds beta-arr2 in a transient manner (class A GPCR), and this binding is required for the agonist-induced internalization of the receptor. In contrast, constitutive CB1R internalization is independent of the beta-arr2 binding of the receptor, suggesting that the molecular mechanisms underlying these two processes are different.