Hypercalcemia is often observed in postmenopausal women as well as in patients with primary hyperparathyroidism or malignant tumors. In this study, we investigated the relationship between calcium ion (Ca²⁺) levels in lacrimal fluid and the rate of corneal wound healing in hypercalcemia using ovariectomized (OVX) rat debrided corneal epithelium. We also determined the effects of Ca²⁺ levels on cell adhesion, proliferation and viability in a human cornea epithelial cell line (HCE-T). The calcium content in bones of OVX rats decreased after ovariectomy. Moreover, the Ca²⁺ content in the blood of OVX rats was increased 1 month after ovariectomy, and decreased. The Ca²⁺ content in the lacrimal fluid of OVX rats was also increased after ovariectomy, and then decreased similarly as in blood. Corneal wound healing in OVX rats was delayed in comparison with Sham rats (control rats), and a close relationship was observed between the Ca²⁺ levels in lacrimal fluid and the rate of corneal wound healing in Sham and OVX rats (y=−0.7863x+8.785, R=0.78, n=25). In addition, an enhancement in Ca²⁺ levels caused a decrease in the viability in HCE-T cells. It is possible that enhanced Ca²⁺ levels in lacrimal fluid may cause a decrease in the viability of corneal epithelial cells, resulting in a delay in corneal wound healing. These findings provide significant information that can be used to design further studies aimed at reducing corneal damage of patients with hypercalcemia.