Lung surfactant is secreted through the fusion of lamellar bodies with the plasma membrane of alveolar epithelial type II cells. Annexin A2, a Ca²⁺- and phospholipid-binding protein, promotes the fusion of lamellar bodies with the plasma membrane. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are known to have an essential role in surfactant secretion. We hypothesized that annexin A2 acts as a Ca²⁺ sensor and mediates membrane fusion via its interaction with SNAREs. Both purified or endogenous annexin A2 in type II cells specifically bound with SNAP-23 in a Ca²⁺-dependent manner as determined by pull-down experiments using recombinant glutathione S-transferase-tagged SNAP-23. A deletion study identified the cysteine-rich region (CRR) of SNAP-23 as the binding site for annexin A2. Mutations of cysteine residues in the CRR dramatically decreased the binding. SNAP-23 also co-immunoprecipitated with annexin A2; however, a SNAP-23 mutant failed to co-immunoprecipitate with annexin A2. Immunofluorescence revealed a co-localization of SNAP-23 and annexin A2 in type II cells. Furthermore, anti-SNAP-23 antibody significantly inhibited annexin A2-mediated fusion between lamellar bodies and the plasma membrane. These data suggest that annexin A2 and SNAP-23 are involved in the same pathway in the regulation of lung surfactant secretion.