Latroeggtoxin‐VI (LETX‐VI) is a peptide neurotoxin discovered from Latrodectus tredecimguttatus eggs. In the current study, the action features of the neurotoxin on PC12 cells were systematically investigated. LETX‐VI could promote dopamine release from PC12 cells in the absence and presence of Ca2+, involving an even more complex action mechanism in the presence of Ca2+ and when the treatment time was longer. Although LETX‐VI enchanced the autophagy and secretion activity in PC 12 cells, it showed no remarkable influence on the proliferation, cell cycle, apoptosis and ultrastructure of the cells. Pulldown combined with CapLC‐MS/MS analysis suggested that LETX‐VI affected PC12 cells by interacting with multiple proteins involved in the metabolism, transport, and release of neurotransmitters, particularly dopamine. The low cytotoxicity and effective regulatory action of LETX‐VI on PC12 cells suggest the potential of the active peptide in the development of drugs for the treatment of some dopamine‐related psychotic diseases and cancers.