During neurotransmitter release, exocytosed neurotransmitter vesicles are recycled by endocytosis, which involves the assembly of a complex of endocytic proteins. Assembly of endocytic proteins into a functional complex depends on their dephosphorylation by calcineurin, a calcium-sensitive protein phosphatase and the inhibitory target of immunosuppressive drugs cyclosporin A and FK506. Cain is a recently identified protein inhibitor of calcineurin. We now provide evidence that cain is a component of the endocytic protein complex. The proline-rich region of cain forms a stable association with the SH3 domain of amphiphysin 1. Using a transferrin uptake assay, we found that overexpression of cain in HEK293 cells blocks endocytosis as potently as expression of a dominant negative dynamin 1 construct. The use of other calcineurin inhibitors such as cyclosporin A and FK506 also blocks endocytosis. Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, our results suggest that cain negatively regulates synaptic vesicle endocytosis by inhibiting calcineurin activity, rather than sterically interfering with the assembly of the endocytic protein complex.