<p>Selective serotonin reuptake inhibitors (SSRIs) are characterized by delayed therapeutic onset largely due to their reliance on the desensitization of 5-HT_1A autoreceptors (5-HT_1AR_autos) within the dorsal raphe nucleus (DRN). It has been shown that dissociation of serotonin transporter (SERT) and neuronal nitric oxide synthase (nNOS) interaction selectively modulates 5-HT_1AR_autos, thereby facilitating fast-onset antidepressant responses. Targeting the atypical disk large/ZO-1 (PDZ) domain has been implicated in the SERT-nNOS interaction. In this study, we established a drug screening system based on mBRET combined with biological tests to find SERT-nNOS interaction blockers (SNIBs). During screening the compound libraries, 9 top candidates were found to be capable of binding to the PDZ domain of nNOS. We then identified esflurbiprofen as a promising fast-onset antidepressant candidate. Pharmacodynamic studies revealed that esflurbiprofen effectively penetrated the DRN following systemic administration. Esflurbiprofen (10, 20, 40 mg/kg, i.p., once every 4 days) dose-dependently ameliorated depressive-like behaviors in mice subjected to chronic social defeat stress (CSDS) and chronic restraint stress (CRS). In rs-fMRI analysis, we found that esflurbiprofen enhanced the functional connectivity of emotion-related neural networks in CSDS mice. We further demonstrated that esflurbiprofen disrupted the SERT-nNOS complex in the DRN, augmented membrane-associated SERT, and reduced the concentration of 5-HT in the extracellular space of the DRN. This cascade subsequently enhanced serotonergic neuronal firing through the inhibition of negative feedback mediated by 5-HT_1AR_autos, culminating in an augmented release of 5-HT from serotonergic neurons projecting to the prefrontal cortex and hippocampus. These results highlight the potential of esflurbiprofen to induce rapid antidepressant effects by targeting the SERT-nNOS interaction within the DRN.</p>