We have recently reported that cannabinoid agonists can upregulate and enhance the activity of 5-HT_2A receptors in prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2Areceptors has been associated with neuropsychiatric disorders such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively upregulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist induced upregulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced upregulation of GRK5 involves CB₂ receptors, β-Arrestin 2 and ERK1/2 signaling as treatment with CB₂ shRNA lentiviral particles, β-Arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented thecannabinoid agonist induced upregulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist- induced upregulation and enhanced 5-HT_2A receptor mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-Arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which upregulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-Arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.