Botulinum neurotoxins (BoNTs) are among the most potent protein toxins known to humans, yet the intracellular trafficking of BoNT-Light Chain A1 (LC/A1) to its substrate, Synaptosome Associated Protein of 25-kDa (SNAP-25), remains poorly understood. A mouse neuroblastoma-2A (N2A) cell-based assay was used to track the intracellular trafficking of cytosolic EGFP-tagged LC/A1. The results revealed that LC/A1 associates with intracellular vesicles as a function of the LC/A1-N terminus, while plasma membrane binding is facilitated by an internal region of LC/A1 that can target LC/A1 to the plasma membrane from the cytosol. An internal LC/A domain, termed the Membrane Localization Domain (MLD), is responsible for the movement of the LC/A1 to the plasma membrane, where the association may be stable or reversible depending on the LC/A subtype. Recent studies have detected the physical trafficking of EGFP-LC/A1 from the cytosol to the intracellular plasma membrane. Thus, in addition to known steps such as host receptor binding and catalysis, the intracellular trafficking of LC to the target substrate may be a critical determinant of BoNT potency. Mechanistic insights into BoNT intracellular trafficking will clarify fundamental aspects of toxin action towards the development of novel therapeutic strategies to mitigate BoNT toxicity.