SH3 protein interacting with Nck, 90 kDa (SPIN90) is an Nck-binding protein that contains one Src homology 3 (SH3) domain, three proline-rich domains (PRDs), a serine/threonine-rich region, and a hydrophobic C-terminal region. Previously, we have shown that SPIN90 plays roles in the sarcomere assembly in cardiac muscles and in the formation of focal contacts in HeLa cells. Besides in heart, SPIN90 is also highly expressed in the brain, but its role in the neuronal system is completely unknown. Here, we found that SPIN90 is expressed in the presynaptic compartment in which it binds dynamin I, a key component of the endocytic machinery, and that it participates in synaptic vesicle endocytosis. Pull-down analysis and coimmunoprecipitation proved the associations of SPIN90 with dynamin I through SH3-PRD interaction. Overexpression of SPIN90 or knocking down SPIN90 by small interfering RNA impaired synaptic vesicle endocytosis. We further confirmed by the rescue experiments that the endocytic defects by SPIN90 expression come from its interaction with dynamin I. Exocytosis kinetics was not affected by SPIN90 expression. Together, our findings suggest that SPIN90 could modulate the interactions of dynamin I with other endocytic proteins that cooperate in the coated vesicle formation, thus regulating synaptic vesicle endocytosis.