Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP₇) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP₇ action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP₇ levels, caused by overexpression of inositol hexakisphosphate (IP₆) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP₇ concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP₇, but not 1-IP₇, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP₆. Synaptotagmin 1 (Syt1), a Ca²⁺ sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP₇. Notably, 5-IP₇ showed a 45-fold higher binding affinity for Syt1 compared with IP₆. In addition, 5-IP₇–dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca²⁺ levels. Thus, 5-IP₇ appears to act through Syt1 binding to interfere with the fusogenic activity of Ca²⁺. These findings reveal a role of 5-IP₇ as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.