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Recent studies have shown that soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in exocytotic release in mast cells as in neurotransmitter release. However, the roles of the proteins that regulate the structure and activity of SNARE proteins are poorly understood. Complexin is one such regulatory protein and is involved in neurotransmitter release, although ideas about its role are still controversial. In this study, we investigated the expression and role of complexin in the regulation of exocytotic release (degranulation) in mast cells. We found that complexin II, but not complexin I, is expressed in mast cells. We obtained RBL-2H3 cells that expressed a low level of complexin II and found that antigen-induced degranulation was suppressed in these cells. No significant changes in the Ca2+ response or expression levels of syntaxins and synaptotagmin were observed in knockdown cells. An immunocytochemical study revealed that complexin II was distributed throughout the cytoplasm before antigen stimulation. However, the distribution of complexin II changed dramatically with stimulation and it became localized on the plasma membrane. This change in the intracellular distribution was observed even in the absence of extracellular Ca2+, while exocytotic release was inhibited almost completely under this condition. The degranulation induced by phorbol 12-myristate 13-acetate and A23187 depended on the extracellular Ca2+ concentration, and its sensitivity to Ca2+ was decreased in knockdown cells. These results suggest that complexin II regulates exocytosis positively by translocating to the plasma membrane and enhancing the Ca2+ sensitivity of fusion machinery, although this translocation to the plasma membrane is not sufficient to trigger exocytotic membrane fusion.