The striatum is the predominant input structure of the striatum, receiving converging glutamatergic and GABAergic inputs from cortical and subcortical regions. It also receives modulatory input from midbrain dopamine neurons which has been shown to be critical for motor initiation, learning and reward. Additionally, the striatum also receives serotonergic input with one third of serotonergic neurons projecting from the dorsal raphe nucleus (DRN) to the striatum. Here, we studied the impact of 5-HT on the interactions between Cholinergic interneurons (ChINs). ChINs are key modulators of striatal function. Through inhibitory control on medium spiny neurons, ChINs can influence action selection and transmission of reinforcement-related information. ChINs also form polysynaptic connections between each other, enabling them to elicit a concerted signal across large areas of the striatum. Using simultaneous whole-cell recordings of ChINs and pharmacological manipulation, we show that this population of interneurons is responsive to serotonin (5-HT). Serotonin application altered the intrinsic properties of ChINs and attenuated polysynaptic inhibition among them. Addition of 5-HT2 receptor antagonist, ketanserin, demonstrated that these responses were 5-HT2 receptor dependent. These data present a novel way by which networks of ChINs are modulated by serotonin, thus adding to the complexity of interactions between acetylcholine, dopamine, and serotonin in striatal circuit modulation. Due to the broad effects of acetylcholine release in the striatum, serotonin likely has far-reaching effects on striatal subtypes and inputs, which may provide insight into the mechanisms underlying non-motor symptoms of Parkinson’s Disease.