Synaptotagmins (SYT) are calcium-binding proteins that participate in regulated exocytosis. Although SYTI to IX isoforms are expressed in insulin-producing cell lines, hitherto only SYTIX has been associated with native -cell insulin granules and implicated in exocytosis. SYTVII was also proposed to regulate insulin exocytosis, but its subcellular location and number of alternative splice variants produced remain controversial. Only transcripts of SYTVII , , and a novel splice variant are expressed in -cells and INS-1E cells. Western blotting revealed that INS-1E cells predominantly produced SYTVII and low levels of SYTVII , whereas SYTVII was undectable. The protein colocalized with insulin granules but not with synaptic-like microvesicles. Overexpression of SYTVII resulted in decreased insulin granule content with a concomitant translocation of the variant to the plasma membrane, while SYTVII retained largely a granular pattern. Overexpressed SYTVII exhibited a distribution different to that of insulin granules and inhibited exocytosis when assessed by whole cell patch clamp capacitance recording. Silencing of SYTVII by targeted RNA interference suppressed secretion, while repression of slightly increased release. Our results demonstrate that SYTVII is expressed on insulin granules and that only SYTVII is implicated in exocytosis under physiological conditions.—Gauthier, B. R., Duhamel, D. L., Iezzi, M., Theander, S., Saltel, F., Fukuda, M., Wehrle-Haller, B., Wollheim. C. B. The synaptotagmin VII splice variants , , and are expressed in pancreatic -cells and regulate insulin exocytosis