Insulin secretion is precisely regulated by blood glucose With unique biphasic pattern. The regulatory mechanism of the second-phase insulin release is unclear. In this study, we report that DOC2b (double C2 domain protein isoform b), a SNARE related protein, was associated with insulin vesicles and translocated to plasma membrane within several minutes upon high-glucose stimulation followed by an interaction with syntaxin4, but not syntaxin1. This binding specificity and the time Course of DCC2b translocation were Suitable for the regulation of second-phase insulin release. Increased DOC2b expression enhanced glucose-stimulated insulin secretion. In contrast, silencing DOC2b inhibited delayed release of insulin, Without affecting rapid (similar to 7 min) phase secretion. Interestingly, DOC2b had no effects on KCl-triggered insulin release. These data suggest that DCC2b may be a regulator for delayed (second-phase) insulin secretion in MIN6 cells. (C) 2009 Elsevier Inc. All rights reserved.