Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that blocks synaptic transmission via the cleavage of SNAP-25 (synaptosomal-associated protein of 25 kDa). BoNT/A is successfully used in clinical neurology for the treatment of several neuromuscular pathologies and pain syndromes. Despite its widespread use, relatively little is known on BoNT/A intracellular trafficking in neurons. Using the visual pathway as a model system, here we show that catalytically active BoNT/A is capable of undergoing anterograde axonal transport and transcytosis. Following BoNT/A injection into the rat eye, significant levels of BoNT/A-cleaved SNAP-25 appeared in the retinorecipient layers of the superior colliculus (SC). Anterograde propagation of BoNT/A effects required axonal transport, ruling out a systemic spread of the toxin. Cleaved SNAP-25 was present in presynaptic structures of the tectum, but retinal terminals were devoid of the immunoreactivity, indicative of transcytosis. Experiments based on sequential administration of BoNT/A and BoNT/E showed a persistent catalytic activity of BoNT/A in tectal cells following its injection into the retina. Our findings demonstrate that catalytically active BoNT/A is anterogradely transported from the eye to the SC and transcytosed to tectal synapses. These data are important for a more complete understanding of the mechanisms of action of BoNT/A.