Hippocampal neurons in the brain polarize to form multiple dendrites and one long axon. The formation of synaptic connections requires precise coordination of pre- and postsynaptic neurons. Here, we report that the overexpression of presynaptic Synaptotagmin1, an abundant and important presynaptic vesicle protein that binds Calcium (Ca2+), leads to abnormal multiple axon formation in cultured rat hippocampal neurons. Ca2+ influx into presynaptic nerve terminals activates synaptic vesicle exocytosis by triggering fast synchronous fusion and a slower asynchronous release pathway. A brief rise in Ca2+ after consecutive action potentials has been correlated with a form of short-term synaptic plasticity with enhanced vesicle fusion, termed facilitation. Synaptotagmin1 was originally identified as an abundant synaptic vesicle protein that binds Ca2+ and phospholipids. It is now widely thought to be the major Ca2+ sensor for neurotransmitter release in all species from lower invertebrates to mammals. The potential role of synaptotagmin1 in neuronal development, including axon differentiation, remains unclear. We determined this by using and checking anti-Tau1 and anti-sodium channel (anti-NaCh) as marker axons. We also found that the overexpression of Synaptotagmin1 in hippocampal neurons triggered an increase in the number of dendrites. To further dissect the functions of endogenous Synaptotagmin1 in neuronal polarity, we used the shRNA of Synaptotagmin1 that specifically blocks the existence of endogenous Synaptotagmin1. When the shRNA of Synaptotagmin1 was introduced to the cells, the number of axons and dendrites did not change. These results indicate that the accumulation of Synaptotagmin1 may play an important role in axon/dendrite differentiation.