Recent studies have shown that the zona incerta (ZI) plays a key role in modulating defensive behaviors. Accumulation of evidence in the lab has revealed that stress or potential threat engages substantia nigra-to-zona incerta (SN-to-ZI) input to promote defensive behavior. Nevertheless, how the SN-to-ZI input recruits its downstream regions to regulate defensive behavior remains unclear. To address this question, we examine the functional recruitment of SN-to-ZI input by combining excitatory optogenetic manipulation with neural activity marker c-Fos staining to map involved downstream brain regions that may mediate the regulation of SN-to-ZI input on defensive behavior. Counting results indicated that, comparing to the control group, there were significantly more c-Fos-expressing cells detected in the lateral habenula (LHb) and lateral periaqueductal gray (lPAG) when the SN-to-ZI input was optogenetically activated. We further employed both anterograde and retrograde tracing approaches to examine SN-innervated ZI projections to the LHb and LPAG, since both regions are well known for their roles in regulating defensive behaviors. Colocalization analyses indicated that ~39% and ~22% of SN-innervated ZI cells respectively sent axonal projections to the LHb and LPAG. On the other hand, ~74% of LHb-projecting and ~39% of PAG-projecting ZI cells received SN innervations. Further investigation will be required to examine the causal roles of these downstream brain regions in mediating defensive behaviors.