<p>Solute carrier family member 10A4 (SLC10A4) is an orphan transporter selectively expressed in aminergic cells, where it localizes to synaptic vesicles and has been implicated in aminergic neurotransmission. Although constitutive knockout studies have suggested a role for SLC10A4 in monoamine and acetylcholine homeostasis, interpretation has been limited by developmental compensation and lack of cell-type specificity. Here, we employed a tamoxifen-inducible CreERT2–lox system to selectively delete <i>Slc10a4</i> in serotonergic neurons of young adult mice (8-10 weeks), enabling assessment of its function in mature serotonergic circuits. Adult serotonergic deletion of SLC10A4 resulted in a marked reduction of baseline extracellular serotonin levels in the medial prefrontal cortex, as measured by in vivo microdialysis. Notably, the ability of the selective serotonin reuptake inhibitor fluoxetine to elevate extracellular serotonin was strongly attenuated. These neurochemical deficits were accompanied by increased anxiety-like behavior across multiple behavioral paradigms and a blunted antidepressant-like response to fluoxetine in the forced swim test, while locomotor activity and motor coordination remained intact. These findings show that SLC10A4 is required for normal extracellular serotonin availability and antidepressant responsiveness in the adult brain. Together, our results identify SLC10A4 as a novel critical regulator of serotonergic neurotransmission and suggest that presynaptic monoamine handling represents an important and previously underappreciated determinant of affective behavior and treatment efficacy.</p>