The subunit B of cholera toxin (CTB), which specifically binds with ganglioside GM1 enriched in membrane lipid rafts, is known to interfere with multiple cell functions. However, the specific, stable and spatially defined membrane signaling induced by CTB binding is often difficult to investigate by applying CTB molecules in bulk solution due to quick internalization, elicited intracellular reactions, and homogeneous interaction with the entire cell membrane. Here, we interfaced the neuroendocrine PC12 cells with surface immobilized and patterned CTB molecules, and interrogated the effects of CTB binding on vesicular exocytosis using integrative single-cell study methods. It was discovered that CTB binding facilitates vesicle trafficking, docking and exocytosis in a cholesterol dependent manner. And these effects are probably attributable to the increased membrane GM1 and cholesterol, and enhanced Ca(2+) signaling.