Beyond the renowned roles in motivation regulation, recent wave of studies has revealed that the ventral tegmental area (VTA) and substantia nigra (SN) are also involved in regulating defensive behaviors. More recently, the zona incerta (ZI), an overlooked midbrain downstream and functionally mysterious inhibitory nucleus, has been reported to play a role in modulating defensive behaviors. Despite that the ventral midbrain sends projection to the ZI, the neurochemical phenotypes of cells involved in the connection remain poorly understood.Thus we employed retrograde and anterograde tracing approaches in the brains of Ai14 reporter mice to respectively target ZI-projecting ventral midbrain cells and SN-innervated ZI cells. By conducting RNAscope in situ hybridization to characterize neurochemical phenotypes, we first uncovered a biased retrograde labeling toward non-DA neurons in the ventral midbrain by employed retroAAV. The colocalization analysis indicated that the major subpopulation of ZI-projecting ventral midbrain cells were GABAergic neurons. Interestingly, GABAergic neurons also accounted for ~60% of the SN-innervated ZI cells. We further examined the downstream projections of ZI cells that were innervated by the SN. We employed an intersectional anterograde tracing approach to target SN-innervated ZI cells. Following robust viral expression along the axons, we mapped the EYFP-expressing axonal projections of SN-innervated ZI cells. Prominent innervations were detected in several regions, including lateral habenula (LHb) and lateral periaqueductal gray (LPAG), which are well known for their roles in regulating defensive behaviors. These results suggest that SN-to-ZI input may be likely through a disinhibiting mechanism to engage neural substrates of defense.