Abstract The periaqueductal gray (PAG) is implicated in numerous defensive responses such as analgesia, freezing and avoidance. However, which subdivisions and cell types within the PAG are responsible for these behaviors is not completely understood. Cholecystokinin-expressing neurons (CCK+) robustly populate the lateral/ventrolateral PAG, yet they remain largely uncharacterized. Our early fiber photometry recordings indicate higher activity in CCK+ l/vlPAG neurons correlates with safety seeking. To probe the role of these neurons in safety seeking, we developed a Latency To Enter assay in which a mouse is placed in a box that contains a burrow (a dark safe space), and the latency (the time taken to enter the burrow) is measured. A lower latency indicates a stronger safety seeking response. Optogenetic activation and inhibition of CCK+ l/vlPAG neurons significantly reduces and increases latency to enter respectively, indicating activity in these neurons is sufficient and necessary for safety seeking. Together, these results exhibit the role of CCK+ l/vlPAG neurons in safety seeking, mapping a specific adaptive response to a genetically-defined PAG neuronal population.