The midbrain periaqueductal gray (PAG) coordinates the expression and topography of defensive behaviors to threat and also plays an important role in Pavlovian fear learning itself. Whereas the role of PAG in the expression of defensive behavior is well understood, the relationship between the activity of PAG neurons and fear learning, the exact timing of PAG contributions to learning during the conditioning trial, and the contributions of different PAG columns to fear learning are poorly understood. We assessed the effects of optogenetic inhibition of lateral (LPAG) and ventrolateral PAG (VLPAG) neurons on fear learning. Using adenoassociated viral vectors expressing halorhodopsin, we show that brief optogenetic inhibition of LPAG or VLPAG during delivery of the shock unconditioned stimulus (US) augments acquisition of contextual or cued fear conditioning, and we also show that this inhibition augments postencounter defensive responses to a nonnoxious threat. Taken together, these results show that LPAG and VLPAG serve a key role in the regulation of Pavlovian fear learning at the time of US delivery. These findings provide strong support for existing models that state that LPAG and VLPAG contribute to a fear prediction error signal determining variations in the effectiveness of the aversive US in supporting learning.