We previously demonstrated that botulinum neurotoxin A (BoNT-A) exerts bilateral antinociceptive effects, involving trans-synaptic transport at the level of the lumbar spinal cord. However, the potential distribution of the toxin to supraspinal sites has not yet been investigated. In the present study, we examined the distribution of cleaved SNAP-25 (cl-SNAP-25), a marker of BoNT-A activity, in the rat brain following peripheral unilateral BoNT-A administration. Brain tissues from rats treated with BoNT-A (7 U/kg, into the hind paw) were analyzed using immunofluorescent tyramide signal amplification to detect cl-SNAP-25. To assess the contribution of trans-synaptic transport, a BoNT-A-neutralizing antitoxin (2 IU) was administered intrathecally 24 h after BoNT-A injection. Signal intensity was evaluated using a semi-quantitative immunohistochemical scoring method based on cl-SNAP-25-positive nerve fibers. Bilateral cl-SNAP-25 immunoreactivity was observed in multiple supraspinal regions, most prominently within the trigeminal complex and the facial and gracile nuclei. Signal intensity was significantly reduced by intrathecal antitoxin, indicating that trans-synaptic transport contributes to central BoNT-A distribution. Peripherally administered BoNT-A reaches distant supraspinal regions, possibly via neuronal retrograde and trans-synaptic transport. Further studies are warranted to clarify exact pathways and alternative distribution routes, determine the functional relevance of central BoNT-A presence, and assess its clinical implications.