Dysregulation of serotonin (5-HT) transmission has been implicated as a factor in the development of depression. This study determined the effects of Tat on serotonin transporter (SERT), dynamic 5-HT release, and the effect of induction of Tat expression on mobility in the tail-suspension test (TST) in inducible Tat transgenic (iTat-tg) mice. Optimization of recombinant Tat1-86 (rTat1-86) concentration revealed a Ki of 7.2 nM for it inhibiting 5-HT uptake into mouse synaptosomes. SRI-32743 displayed a partial antagonism with 13 nM affinity and 69.5% Emax for inhibiting 5-HT uptake in CHO cells transfected human SERT (hSERT). SRI-32743 (5, 50, 500 nM) did not alter the Vmax of [3H]5-HT uptake in hSERT. Moreover, 8.75 nM rTat1-86 inhibited 5-HT uptake by 40% in hSERT and 25% in mouse synaptosomes, which was attenuated by SRI-32743 (50, 1 nM, respectively). Fast scan cyclic voltammetry showed that 14-day doxycycline (Dox)-treated iTat-tg mice displayed an increase in the baseline 5-HT release in substantia nigra relative to G-tg (Tat null) mice. Addition of escitalopram exacerbated Tat’s effect on 5-HT release. Dox treated iTat-tg mice displayed a 116% decrease and 147% increase of the Vmax of [3H]5-HT uptake in the midbrain and the prefrontal cortex, respectively. SRI-32743 (1 mg/kg/d, i.p.) alleviated Tat-potentiated immobility in TST in 14-day Dox treated iTat-tg mice compared to saline control. These results provide mechanistic insights and reveal the potential for the therapeutic use of allosteric modulators for mitigating depressive behaviors in HIV positive individuals.