Botulinum neurotoxin serotype A (BoNT/A) is a versatile protein therapeutic used to treat a variety of neuromuscular and pain disorders. BoNT/A is a 150 kDa protein consisting of three domains, including a 50 kDa receptor binding domain (HC/A). To enter neurons, the HC/A binds a receptor complex composed of proteins and gangliosides. Members of the synaptic vesicle (SV2) glycoprotein family were reported to mediate BoNT/A uptake by facilitating toxin binding and internalization during vesicle recycling in depolarized neurons. Previously, we identified Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity component of BoNT/A receptor complex. The interaction between FGFR3 and recombinant HC/A (rHC/A) was analyzed using Cross Correlation RICS (ccRICS). Differentiated SiMa, PC-12, and SH-SY5Y cells expressing a fluorescent FGFR3-Halo tag chimera were treated with Alexa-Fluor®633-rHC/A. These proteins move together on the membrane of SiMa and PC-12 cells with a diffusion rate of 3.2 μm2/s and after vesicular endocytosis, the rHC/A-FGFR3 complex diffuses at a rate of 1.1 μm2/s. The GccRICS (0, 0) of rHC/A and FGFR3 peaks in SiMa, PC-12, and SH-SY5Y after 2, 6, and 9 hours treatment, respectively. We investigated the trafficking of rHC/A in PC-12 cells with GFP labeled markers for early endosomes and synaptic vesicles. The fluorescent signal from rHC/A cross correlated with early endosomes when the cells were treated in basal or depolarizing media. However, cross-correlation with synaptic vesicles was only observed when the cells were depolarized. These data suggest that BoNT/A can enter neurons via the endosomal and/or synaptic vesicle pathways depending on the cell's polarization state. FGFR3 is part of a high-affinity receptor complex for BoNT/A in neuronal membranes and the FGFR3-BoNT/A interaction plays a role in productive trafficking of the toxin in neuronal cells.