Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely-soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and plasma membrane. Upon activation of the sympatho-adrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of co-packaged peptide hormones. Thus, regulated expansion of the secretory fusion pore is a control point for differential-hormone release of the sympatho-adrenal stress response. Previous work has shown syndapin 1 deletion to alter transmitter release and dynamin 1/syndapin 1 interaction as necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contribute to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin/syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by the mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.