The 5-HT6 receptor (5-HT6R) plays roles in cognition, anxiety and learning and memory disorders, yet new details concerning its regulation remain poorly understood. In this study, we found that 5-HT6R directly interacts with SNX14 and this interaction dramatically increases internalization and degradation of 5-HT6R. Knockdown of endogenous SNX14 has the opposite effect. SNX14 is highly expressed in the brain and contains a putative regulator of G-protein signaling (RGS) domain. Although its RGS domain is found to be non-functional as a GTPase activator for Gαs, we found that it specifically binds and sequesters Gαs, thus inhibiting downstream cAMP production. We further found that PKA-mediated phosphorylation of SNX14 inhibited its binding to Gαs and diverted SNX14 from Gαs binding to 5-HT6R binding, thus facilitating the endocytic degradation of the receptor. Therefore, our results suggest that SNX14 is a dual endogenous negative regulator in 5-HT6R-mediated signaling pathway, modulating both signaling and trafficking of 5-HT6R.