The basal ganglia are an evolutionarily old group of structures, with gross organization conserved across species. Despite this conservation, there is evidence suggesting that anatomical organization of a key output nucleus of the basal ganglia, the substantia nigra pars reticulata (SNpr), diverges across species. Nevertheless, there are relatively few comparative studies examining the impact of manipulations of SNpr across species. Here, we evaluated the role of SNpr in a highly conserved behavior: prepulse inhibition of the acoustic startle response (PPI). We performed parallel experiments in both rats and rhesus macaques using intracranial microinfusions of GABAA agonist muscimol to investigate the role of SNpr in PPI. SNpr inactivation significantly disrupted PPI in rats, congruent with prior studies; however, in macaques, SNpr inactivation resulted in facilitation of PPI. We suggest that this difference in circuit function results from a divergence in anatomical connectivity, underscoring the importance of circuit dissection studies across species.