Background: Proparacaine hydrochloride (PH), as a a local anesthetic, is used regularly in ophthalmic surgery and optometry. However, few pieces of research on the ocular surface toxicity of PH eye drops have so far been reported. Purpuse: To evaluate the effect of PH on the ocular surface of mice. Methods: Male C57/BL6 mice were divided into four groups: normal, vehicle-treated, 0.05% PH-treated and 0.5% PH treated 7 days. The clinical indications were tear production, corneal sensitivity, and fluorescein staining. The structure and morphology of the cornea were examined by confocal microscopy; the thickness of the corneal center was examined by anterior segment optical coherence tomography (AS-OCT); and the corneal epithelial microvilli morphology was examined using scanning electron microscopy. Hematoxylin-eosin staining was used to characterize the central corneal morphology. Immunofluorescence staining for cytokeratin 10 (K10) was employed to evaluate squamous metaplasia in the corneal epithelium. Results: The results show that topical PH treatment diminished tear production and corneal sensitivity, and increased corneal fluorescein staining scores. Moreover, PH altered the corneal epithelial microvilli morphology, disrupted the epithelial barrier of the cornea, and promoted apoptosis of ocular surface cells. Furthermore, the expression of K10 in the corneal epithelium was found to be increased. Conclusion: Treatment with 0.5% PH caused instability of the tear film, and changes in corneal sensitivity and ocular surface homeostasis.