Introduction Richardson's ground squirrels use alarm calls to warn conspecifics about potential predatory threats. Chirp calls typically indicate high levels of threat from airborne predators, while whistle calls are associated with lower levels of threat from terrestrial predators. These types of calls primarily elicit escape behaviors and increased vigilance in receivers, respectively. While much is known about the neural mechanisms involved in the production of vocalizations, less is known about the mechanisms important for the perception of alarm calls by receivers, and whether changes in perceived risk are associated with unique patterns of neuronal activation. Thus, to determine whether alarm calls associated with different levels of predation risk result in differential neuronal activation, we used immunohistochemistry to identify and quantify c-Fos immunopositive cells in brain regions important in stress, fear, danger, and reward, following alarm call reception. Methods We exposed 29 female Richardson's ground squirrels (10 control, 10 whistle receivers, and 9 chirp receivers) to playbacks of whistles, chirps, or a no-vocalization control. We then assessed neuronal activation via c-Fos immunohistochemistry in 12 brain regions. Results Ground squirrels receiving high-threat “chirp” vocalizations had reduced neuronal activation in the medial amygdala and superior colliculus compared with controls. It is likely that changes in activity in these brain regions serve to alter the balance between approach and avoidance in turn promoting escape behaviors. Conclusions Thus, we conclude that in Richardson's ground squirrels, these brain regions are important for the perception of risk resulting from receiving alarm calls and allow for appropriate behavioral responses by receivers.