The ability to recognize and anticipate potential danger is crucial for survival across species. The midbrain periaqueductal gray (PAG) is implicated in regulating threat-anticipatory responses, including heart rate deceleration (threat bradycardia) which is typically observed during freezing. Although animal studies have provided causal evidence that the PAG regulates threat-anticipatory bradycardia, causal evidence does not exist in humans. To address this translational gap, we performed a single-case study to elucidate the causal role of the human PAG in threat-anticipatory bradycardia using deep brain stimulation (DBS). We report on a participant who received PAG DBS for chronic pain treatment. The participant performed an instructed fear task during which cues were presented signaling either threat of electrical shock or safety. During the task, we applied DBS in the PAG, no DBS, or DBS at a control site bordering the PAG. Deep brain stimulation in the PAG significantly increased threat bradycardia responses (i.e. reduced heart rate for threat vs. safety) compared to no stimulation, whereas control-site stimulation bordering the PAG did not significantly affect threat bradycardia. Together, this single-case report provides causal evidence that the human PAG regulates threat bradycardia responses, furthering our understanding of the neural circuit underlying defensive reactions in humans.