The serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT) controls serotonergic neurotransmission in the brain by rapid clearance of 5-HT from the synaptic cleft into presynaptic neurons. SERTs are primary targets for antidepressants for therapeutic intervention of mood disorders. Our previous studies have identified the involvement of several signalling pathways and protein kinases in regulating SERT function, trafficking and phosphorylation. However, whether Akt/PKB (protein kinase) regulates SERT function is not known. In the present study, we made the novel observation that inhibition of Akt resulted in the down-regulation of SERT function through the regulation of SERT trafficking and phosphorylation. Akt inhibitor Akt X {10-(4'-[N-diethylamino)butyl]-2-chlorophenoxazine} reduced the endogenously phosphorylated Akt and significantly decreased 5-HT uptake and 5-HT-uptake capacity. Furthermore, SERT activity is also reduced by siRNA down-regulation of total and phospho-Akt levels. The reduction in SERT activity is paralleled by lower levels of cell-surface SERT protein, reduced SERT exocytosis with no effect on SERT endocytosis and accumulation of SERT in intracellular endocytic compartments with the most prominent localization to late endosomes and lysosomes. Akt2 inhibitor was more effective than Akt1 inhibitor in inhibiting SERT activity. Inhibition of downstream Akt kinase GSK3α/β (glycogen synthase kinase α/β) stimulates SERT function. Akt inhibition leads to a decrease in SERT basal phosphorylation. Our results provide evidence that Akt regulates SERT function and cell-surface expression by regulating the intracellular SERT distribution and plasma membrane availability, which perhaps may be linked to SERT phosphorylation state. Thus any changes in the activation of Akt and/or GSK3α/β could alter SERT-mediated 5-HT clearance and subsequently serotonergic neurotransmission.