Neuronal circuits located in the midbrain play a critical role in controlling defensive behavior. However, it is still elusive how different neuron types contribute to distinct behavioral outcomes during the presence of threat. In this study, we investigated neurons expressing vasoactive intestinal polypeptide (VIP) in the ventral periaqueductal gray (PAG) and dorsal raphe nucleus in mice. These VIP-expressing neurons also expressed tyrosine hydroxylase (TH), a marker for dopaminergic cells. Using viral tracing conducted in Vip-Cre mice, we observed that these VIP-expressing neurons innervated exclusively the bed nucleus of stria terminalis (BNST) and central amygdala (CeA). Importantly, in human tissue we identified VIP-immunoreactive neurons in the PAG and axons located in the CeA. Using channelrhodopsin 2-assisted circuit mapping, we revealed that the two main neuronal subpopulations in the mouse BNST and CeA, expressing somatostatin and protein kinase C delta, were not the primary postsynaptic targets of PAG VIP neurons. Electron microscopy determined that spines were the major target of VIP-immunoreactive axon terminals in both nuclei. To clarify the role of these midbrain VIP-expressing neurons in fear behavior, we inhibited their activity using chemogenetics and observed that their inhibition during fear conditioning impaired the recall of contextual, but not cued, fear memory tested on subsequent days. These results collectively show that this peptidergic midbrain-extended amygdala pathway, present both in humans and mice, plays a role in the regulation of contextual fear learning.