The effects of serotonin (5-HT) on anxiety and depression are mediated by a number of 5-HT receptors, including autoreceptors that act to inhibit 5-HT release. While the majority of anxiety and depression-related research has focused on the 5-HT1A receptor, the 5-HT1B receptor has a lesser known role in modulating emotional behavior. 5-HT1B receptors are inhibitory GPCRs located on the presynaptic terminal of both serotonin and non-serotonin neurons, where they act to inhibit neurotransmitter release. The autoreceptor population located on the axon terminals of 5-HT neurons is a difficult population to study due to their diffuse localization throughout the brain that overlaps with 5-HT1B heteroreceptors (receptors located on non-serotonergic neurons). In order to study the contribution of 5-HT1B autoreceptors to anxiety and depression-related behaviors, we developed a genetic mouse model that allows for selective ablation of 5-HT1B autoreceptors. Mice lacking 5-HT1B autoreceptors displayed the expected increases in extracellular serotonin levels in the ventral hippocampus following administration of a selective serotonin reuptake inhibitor. In behavioral studies, they displayed decreased anxiety-like behavior in the open field and antidepressant-like effects in the forced swim and sucrose preference tests. These results suggest that strategies aimed at blocking 5-HT1B autoreceptors may be useful for the treatment of anxiety and depression.