Synaptotagmin I or II (Syt I/II) is involved in Ca2+-regulated exocytosis of secretory vesicles, probably serving as a Ca2+-sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca2+-regulated exocytosis. Immunoblots confirmed the high specificity of the anti-STI/II-C2A antibody and showed the expression of synaptotagmin I or II in chick dorsal root ganglion neurons. Immunocytochemistry revealed that synaptotagmin I or II is enriched at the growth cone region of chick dorsal root ganglion neurons, in both lamellipodia and filopodia. Whole or Fab-fragment of the anti-STI/II-C2A antibody loaded into dorsal root ganglion neurons by trituration significantly inhibited neurite outgrowth, whereas preimmune immunoglobulin G had no effect. These results showed that the C2A domain of synaptotagmin I or II plays a crucial role in neurite outgrowth.