To understand the secretory mechanisms and physiological role of insulin in the tear film, the present study examined: (1) the time-course of insulin secretion in the tear film under glucose intravenous stimulation, (2) the glucose- and carbachol-induced insulin secretion from isolated lacrimal gland (LG), (3) the effect of insulin on glucose consumption by the cornea, and (4) the expression of insulin, PDX-1, and glucose transport proteins (GLUT) in lacrimal gland (LG) tissue. The insulin level in the tear film of 8-week-old male Wistar rats increased from 0.6 ± 0.45 to 3.7 ± 1.3 ng/ml in the initial minutes after glucose stimulation. In vitro assays demonstrated that higher glucose concentrations from 2.8 to 16.7 mM, 200 µM carbachol or 40 mM KCl significantly increased insulin secretion from lacrimal glands compared to controls, but did not detect C-peptide as measured by RIA. Glucose consumption by corneal tissue, evaluated by radiolabeled D-[U-¹⁴C] glucose uptake, was 24.07 ± 0.61 and was enhanced to 31.63 ± 3.15 nmol/cornea/2 h in the presence of 6 nM insulin (P=0.033) and to 37.5 ± 3.7 nmol/cornea/2 h in the presence of 11.2 mM glucose (P=0.015). Insulin and PDX-1 mRNA was detected in LG. Insulin was located in the apical areas of acinar cells by immunoperoxidase and the expression of GLUT-1, but not PDX-1, was confirmed by Western blot. These findings suggest that insulin secretion in the tear film is influenced by local stimuli such as nutrient and neural inputs and that this hormone plays a metabolic role in ocular surface tissues. These data also indicate that under normal conditions the insulin secreted by LG is stored, but it is not clear that is locally produced in the LG.