Defensive behaviors against threatening situations are crucial for survival, and maladaptation of neural functions involved in defensive behavior may result in panic-like behavior. Defensive behaviors must be optimized for animals to efficiently avoid danger and maximize their chance of survival. The midbrain periaqueductal gray (PAG) controls defensive behaviors. However, the substrate of dysregulated panic-like defensive responses remains unknown. Using in vivo calcium imaging and recordings in mice, we found that PAG astrocytes are activated during threatening situations and trigger defensive behaviors. Using optogenetic astrocyte modulation and electrophysiological experiments, we provide evidence that PAG astrocyte activation and subsequent ATP release are required for optimal defensive behavior; aberrant activation of PAG astrocytes leads to maladaptive defensive behavior resembling panic-like behavior. Our results suggest that PAG astrocytes are neurobiological substrates underlying defensive dysregulation and might be an important cue in panic-related behaviors via increased calcium activity and ATP release.