To survive, animals have to finely regulate their threat assessment and escape behavior. In mice, the brainstem dorsal periaqueductal gray (dPAG) generates innate defensive behaviors towards a multitude of threats, including predators, prey, and aggressive conspecifics. dPAG integrates threat information coming from diverse inputs with cognitive information coming from cortical areas such as the anterior cingulate cortex (ACC). Glutamatergic layer 5 neurons in ACC project to dPAG where they inhibit the expression of defensive behavior. In vivo single-unit recordings in dPAG identified two major neuron cell types that are modulated during approach to a threat and during escape from that threat, referred to as Assessment+ and Flight+ cells, respectively. However, so far it is not clear if Assessment+ and Flight+ cells correspond to neuron classes that can be distinguished by their connectivity or gene expression patterns, leaving it unclear how they may cooperate to regulate support or trigger defensive behaviors. Here, we performed in vivo single-unit calcium miniscope imaging in Vglut2+ and Vgat+ neurons in dPAG to determine how Assessment+ and Escape+ neurons map to these two populations and whether predator, social, and prey threats recruit the same neurons in dPAG. Moreover, we optogenetically stimulated ACC projections in dPAG, showing that they reduced defensive behavior, and we characterized the expression of glutamatergic and GABAergic markers in ACC target cells in PAG. In summary, this study aims to characterize the activity and function of an ACC to PAG neural circuit in defensive behavior.