Neuroendocrine chromaffin cells release a variety of transmitter molecules into the circulation as a specific function of sympathetic activation. Activity-dependent release of transmitter single species or classes of transmitters is controlled through regulation of the secretory fusion pore. Under sympathetic tone, basal synaptic activity drives chromaffin cells to secrete modest levels of catecholamine through a restricted secretory fusion pore. In contrast, elevated sympathetic firing, as evoked by environmental stress or injury, results in the expansion of the fusion pore for maximal catecholamine release. Pore expansion also facilitates release of co-packaged peptide transmitters from the dense granule core. Therefore, fusion pore expansion is a regulatory step for the activation of the sympatho-adrenal stress response. Despite the physiological impact and importance of this process, the molecular mechanism by which pore expansion is regulated remains unresolved. Here we employ fluorescence imaging in combination with electrophysiological and electrochemical-based measurements to test the role of dynamin I in the regulation of activity-mediated fusion pore expansion in mouse adrenal chromaffin cells (Samasilp et al., 2012). We show that under elevated electrical stimulation, dynamin I is dephosphorylated at Ser-774 by the protein phosphatase, calcineurin. We also demonstrate that disruption of dynamin I binding to syndapin, a step regulated by calcineurin-dependent dynamin dephosphorylation, inhibits fusion pore expansion. Lastly, we show that pharmacologic block of N-WASP function (a syndapin binding partner) limits activity-dependent expansion of the fusion pore. Our results suggest that fusion pore expansion is regulated by a calcineurin-dependent dephosphorylation of dynamin I. Dephosphorylated dynamin I acts via a syndapin/N-WASP signaling cascade, leading to pore expansion.