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Yanwen Zhou,* Ying Liu,* Yunhua Hao, Ya Feng, Lizhen Pan, Wuchao Liu, Bing Li, Libin Xiao, Lingjing Jin, Zhiyu Nie Department of Neurology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China *These authors contributed equally to this work Background: Botulinum neurotoxin type A (BoNT/A) is emerging as a treatment modality for Raynaud’s phenomenon (RP). However, the mechanism of the role of BoNT/A in antagonizing the constriction of arteriola in RP remains unclear. Materials and methods: We tested the constriction of arteriole diameter and the distribution of adrenergic receptors on the rat cremaster modle. Moreover, we measured the secretion of norepinephrine (NE), protein level changes and related receptors on cultured rat superior cervical ganglia neurons(SCGNs), a model of sympathetic neuron. Results: Based on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/Α, prazosin, and BQ123 treatment can result in significant inhibition of arteriole vasoconstriction with the same electrical stimulation. The inhibition effect of prazosin was equivalent to BoNT/A, while BQ123 has a synergistic effect with BoNT/A. After treating SCGNs using BoNT/A for 30 min, the decrease in fluorescence intensity of FM1-43 slowed down which was correlated with the doses of BoNT/A. Furthermore, release of NE in the supernatant was significantly decreased as measured by enzyme-linked immunosorbent assay, 24 h after a high dose of BoNT/A (25 µ/mL). Cleaved-SNAP-25 was detected by western blotting 24 h following BoNT/A (50 µ/mL) treatment. Moreover, receptor SV2C, GM1, and FGFR3 were detected on sympathetic neurons, similarly to cholinergic neurons. Conclusion: Our study showed that BoNT/A could significantly inhibit electrical stimulation-induced arteriole vasoconstriction through the sympathetic pathway. The mechanism was similar to the cholinergic one, in which the vesicle release of sympathetic neurons could be inhibited by cleavage of SNAP-25. The end result was blocked vesicle fusion with the presynaptic membrane after BoNT/A treatment, inhibiting the release of the NE. Keywords: botulinum neurotoxin type A, Raynaud’s phenomenon, α-adrenoceptor, arteriole diameter constrict rate, SNAP-25, SV2C, GM1, FGFR3, sympathetic neuron, vesicle cycle