The activity of the prefrontal cortex is essential for normal emotional processing and is strongly modulated by serotonin (5-HT). Yet, little is known about the regulatory mechanisms that control the activity of the prefrontal 5-HT receptors. Here, we found and characterized a deregulation of prefrontal 5-HT receptor electrophysiological signaling in mouse models of disrupted serotonin transporter (5-HTT) function, a risk factor for emotional and cognitive disturbances. We identified a novel tyrosine kinase-dependent mechanism that regulates 5-HT-mediated inhibition of prefrontal pyramidal neurons. We report that mice with compromised 5-HTT, resulting from either genetic deletion or brief treatment with selective serotonin reuptake inhibitors during development, have amplified 5-HT1Areceptor-mediated currents in adulthood. These greater inhibitory effects of 5-HT are accompanied by enhanced downstream coupling to Kir3 channels. Notably, in normal wild-type mice, we found that these larger 5-HT1Aresponses can be mimicked through inhibition of Src family tyrosine kinases. By comparison, in our 5-HTT mouse models, the larger 5-HT1Aresponses were rapidly reduced through inhibition of tyrosine phosphatases. Our findings implicate tyrosine phosphorylation in regulating the electrophysiological effects of prefrontal 5-HT1Areceptors with implications for neuropsychiatric diseases associated with emotional dysfunction, such as anxiety and depressive disorders.