Intracellular calcium ([Ca²⁺]i) signaling regulates physiological functions in most cells. In secretory organs, such as the pancreas, salivary gland, and lacrimal gland (LG), [Ca²⁺]i elevation in acinar cells triggers fluid secretion, which plays vital roles in the maintenance of functional health across the life-course. It is important to understand the secretory mechanism of secretory organs, but lack of analytic systems available for living animals limits the scope of research to gain deeper insights into the precise mechanism of secretion. We established an intravital imaging system for specific cell types of secretory organs to monitor the [Ca²⁺]i changes using mouse line expressing Yellow Cameleon 3.60, a genetically encoded Ca²⁺ indicator. Elevation of [Ca²⁺]i in specific cell types of secretory organs could be monitored after cholinergic stimulation ex vivo and intravitally. We found that a marked attenuation of LG [Ca²⁺]i response to cholinergic stimulation was induced under pathological conditions by postganglionic denervation. Intravital Ca²⁺ imaging in secretory organs will broaden our understanding of the cellular mechanisms in animal models of secretory diseases.