PURPOSE: The aim of this study was to establish and characterize extraorbital lacrimal gland excision (LGE) as a model of aqueous tear-deficient dry eye disease in mice. METHODS: Female C57BL/6 mice at 6 to 8 weeks of age were randomized to extraorbital LGE, sham surgery, or scopolamine groups. Mice that underwent extraorbital LGE or sham surgery were housed in the standard vivarium. Scopolamine-treated mice were housed in a controlled environment chamber that allowed for the continuous regulation of airflow (15 L/min), relative humidity (30%), and temperature (21-23°C). Clinical disease severity was assessed over the course of 14 days using the phenol red thread test and corneal fluorescein staining. Real-time polymerase chain reaction was performed to assess corneal mRNA expression of interleukin 1β, tumor necrosis factor α, and matrix metalloproteinase 9. Flow cytometry was used to assess T helper cell frequencies in the conjunctivae and draining lymph nodes. RESULTS: Extraorbital LGE markedly reduced aqueous tear secretion as compared with the sham procedure and induced a more consistent decrease in aqueous tear secretion than was observed in mice that received scopolamine while housed in the controlled environment chamber. Extraorbital LGE significantly increased corneal fluorescein staining scores as compared with those of both the sham surgery and scopolamine-treated groups. Extraorbital LGE significantly increased the corneal expression of interleukin 1β, tumor necrosis factor α, and matrix metalloproteinase 9. Further, extraorbital LGE increased T helper 17-cell frequencies in the conjunctivae and draining lymph nodes. CONCLUSIONS: Extraorbital LGE induces aqueous tear-deficient dry eye disease in mice as evidenced by decreased aqueous tear secretion, increased corneal epitheliopathy, and induced ocular surface inflammation and immunity.