Tears are extracellular fluid secreted from the lacrimal gland (LG). Tears consist of a dynamic tri-layered film composed of secretions from the LG, Meibomian gland, and conjunctival goblet cells. The LG secretes the aqueous component of the tear, the Meibomian gland secretes the lipid component, and conjunctival goblet cells secrete mucin. The regulation of LG activity via the autonomic nervous system has been recognized as fundamental to maintaining aqueous tear flow. Here, we describe the role of a hormone, peripheral serotonin, in tear secretion. We found that blood serotonin concentration, changed by feeding a diet deprived of the serotonin precursor tryptophan, correlated with tear secretion, and that a sustained decrease in serotonin resulted in LG atrophy and autophagy. The combination of a decrease in serotonin with the interruption of autonomic neural stimuli to the LG preceded these alterations. Furthermore, we found that the serotonin type 3a receptor expressed in LG acinar cells is involved in tear secretion via intracellular calcium mobilization. Our findings demonstrate that hormonal regulation by serotonin, in cooperation with the autonomic nervous system, regulates tear secretion.