Striatal cholinergic interneurons (CINs) can drive local dopamine release via nicotinic acetylcholine receptors (nAChRs) expressed on dopaminergic axons, but their role in modulating serotonin (5-HT) signaling is poorly understood. Here, we show that synchronous activation of CINs directly triggers local 5-HT release in the dorsal striatum via nAChRs expressed on serotonergic axons. This CIN-5-HT coupling is not detectable in the ventral striatum, despite its substantially denser serotonergic innervation. The nAChR-dependent release not only increases 5-HT levels in the dorsal striatum, but also expands the spatial footprint of serotonergic signaling. In Sapap3 mice, a model of obsessive-compulsive disorder (OCD)-like behavior, this mechanism is exaggerated due to a hypercholinergic state, selectively amplifying the nAChR-dependent component of monoamine release. These findings demonstrate a regionally confined form of acetylcholine-5-HT crosstalk in the striatum and identify CINs as regulators of 5-HT dynamics in both healthy and pathological states.