The zona incerta is important for anxiety-mediated behaviors. It contains a group of dopaminergic cells that coexpress the vesicular GABA transporter, potentially coreleasing dopamine and GABA. The superior colliculus is a known projection site of the zona incerta and supports anxiety-driven behavior. However, it is not known if dopaminergic zona incerta cells innervate the superior colliculus via dopamine and GABA transmission. We expressed channelrhodopsin in zona incerta neurons of Th-cre mice, and their fibres were restricted to the medial motor-related layers of the superior colliculus. The superior colliculus cells received monosynaptic GABA transmission from the zona incerta, and these connected cells were dopamine responsive. These results indicated a direct connection between the zona incerta dopamine-GABA cells and the medial motor layers of the superior colliculus. These results are important in understanding the role of the zona incerta dopamine-GABA cells in the regulation of anxiety-related behaviors in the superior colliculus.