Synaptotagmin-like protein (Slp) 2-a is a putative Rab27A/B-effector protein and is implicated in intracellular membrane transport. However, the precise tissue distribution of Slp2-a protein and its functions remain largely unknown. In this study we used a specific anti-Slp2-a antibody to investigate the tissue distribution of Slp2-a in mice and found that Slp2-a is most abundantly expressed in mouse stomach. Co-immunoprecipitation experiments indicated that Slp2-a interacts with Rab27A/B in vivo. We also discovered that Slp2-a and Rab27A/B are predominantly localized at the apical region of gastric-surface mucous cells, where mucus granules are accumulated. Analysis of Slp2-a mutant mice generated by homologous recombination showed a reduced number of mucus granules, a deficiency of granule docking with the apical plasma membrane in the gastric-surface mucous cells and reduction of mucus secretion by Slp2-a-deficient gastric primary cells. Based on these results, we propose that Slp2-a is part of the mucin secretory machinery in surface mucous cells of mouse stomach.