Serotonin‐1B (5‐HT_1B) autoreceptors are located in serotonin (5‐HT) terminals, along with serotonin transporters (SERT), and play a critical role in autoregulation of serotonergic neurotransmission and are implicated in disorders of serotonergic function, particularly emotional regulation. SERT modulates serotonergic neurotransmission by high‐affinity reuptake of 5‐HT. Alterations in SERT activity are associated with increased risk for depression and anxiety. Several neurotransmitter receptors are known to regulate SERT K_m and V_max, and previous work suggests that 5‐HT_1B autoreceptors may regulate 5‐HT reuptake, in addition to modulating 5‐HT release and synthesis. We used rotating disk electrode voltammetry to investigate 5‐HT_1B autoreceptor regulation of SERT‐mediated 5‐HT uptake into synaptosomes. The selective 5‐HT_1B antagonist SB224289 decreased SERT activity in synaptosomes prepared from wild‐type but not 5‐HT_1B knockout mice, whereas SERT uptake was enhanced after pretreatment with the selective 5‐HT_1B agonist CP94253. Furthermore, SERT activity varies as a function of 5‐HT_1B receptor expression—specifically, genetic deletion of 5‐HT_1B decreased SERT function, while viral‐mediated overexpression of 5‐HT_1B autoreceptors in rat raphe neurons increased SERT activity in rat hippocampal synaptosomes. Considered collectively, these results provide evidence that 5‐HT_1B autoreceptors regulate SERT activity. Because SERT clearance rate varies as a function of 5‐HT_1B autoreceptor expression levels and is modulated by both activation and inhibition of 5‐HT_1B autoreceptors, this dynamic interaction may be an important mechanism of serotonin autoregulation with therapeutic implications. Synapse, 2012. © 2012 Wiley Periodicals, Inc.