Under normal physiological conditions, synaptic vesicle endocytosis is regulated by phosphorylation and Ca²⁺‐dependent dephosphorylation of endocytic proteins such as amphiphysin and dynamin. To investigate the regulatory mechanisms that may occur under the conditions of excessive presynaptic Ca²⁺ influx observed preceding neural hyperexcitation, we examined hippocampal slices following high‐potassium or high‐frequency electrical stimulation (HFS). In both cases, three truncated forms of amphiphysin I resulted from cleavage by the protease calpain. In vitro, the binding of truncated amphiphysin I to dynamin I and copolymerization into rings with dynamin I were inhibited, but its interaction with liposomes was not affected. Moreover, overexpression of the truncated form of amphiphysin I inhibited endocytosis of transferrin and synaptic vesicles. Inhibiting calpain prevented HFS‐induced depression of presynaptic transmission. Finally, calpain‐dependent amphiphysin I cleavage attenuated kainate‐induced seizures. These results suggest that calpain‐dependent cleavage of amphiphysin I inhibits synaptic vesicle endocytosis during neural hyperexcitation and demonstrate a novel post‐translational regulation of endocytosis.