Purpose: To compare inflammatory cytokine and defensin expression in response to experimental dry eye (EDE) in interleukin-1 receptor-1 (IL-1R1)-deficient (KO) mice with age-matched wild-type mice (WT). Methods: EDE was induced by subcutaneous scopolamine injection, exposure to low humidity, and an air draft for 5 days in 4- to 6-week-old KO and WT mice. Expression of cytokines IL-1α, IL-1β, tumor necrosis factor (TNF)-α, IL-6, and mouse β-defensins (mBD)-1, mBD-2, and mBD-3 was evaluated by real-time polymerase chain reaction in scraped corneal epithelial cells and whole conjunctival tissues. A multiplex bead assay was performed to quantitate IL-1α, IL-2, IL-4, IL-10, interferon (IFN)-γ, and TNF-α levels in tear fluid, and an enzyme immunoassay was used to quantitate IL-1β levels in tear fluid. Results: EDE significantly increased RNA transcripts for IL-1α and β in the conjunctiva and for TNF-α in the corneal epithelium of WT mice. Levels of IL-1α, IL-1β, and IL-6 were significantly lower in the corneal epithelium and conjunctiva, and TNF-α was significantly lower in the cornea of KO mice after 5 days of EDE than WT mice. Tear fluid IL-1α concentration increased above baseline on days 2-4 of EDE in WT and KO mice. A similar pattern was observed for tear TNF-α. Tear IL-1β increased throughout the 5 days of EDE in WT and KO mice. IFN-γ, IL-2, IL-4, and IL-10 were undetectable in tear fluid of either strain before or after EDE. Corneal mBD-1 mRNA expression was unchanged and conjunctival mBD-1 transcripts decreased in WT and increased in KO mice with EDE. Untreated WT corneas, but not those of KO mice, expressed mBD-2 transcripts, whereas in the conjunctiva, mBD-2 increased in WT and decreased in KO mice with EDE. Corneal mBD-3 mRNA expression was undetected in WT mice, but increased after EDE in KO mice. Conjunctival mBD-3 transcripts were only detected in WT with EDE. Conclusions: These findings indicate that IL-1 signaling is responsible in part for the increased expression of inflammatory cytokines and the changes in mBDs by the ocular surface tissues in response to desiccating stress. These results show the important regulatory aspects of IL-1 on ocular surface epithelial inflammation.