AIM: To investigate the therapeutic effect of natural extract eye drops containing bee venom, musk, and deer antlers in dry eye disease (DED) animal models. METHODS: Scopolamine-injected DED rats and lacrimal gland-excised rats were allocated into control, saline, and natural extract groups respectively and a normal group (lacrimal gland excision was not performed) in lacrimal gland-excised rats. After eye drop instillation 4 times a day for 5d, corneal fluorescein staining (CFS) scores, tear MUC5AC levels, and tear lactic dehydrogenase (LDH) levels were measured. RESULTS: In scopolamine-injected rats, the natural extract-treated group had significantly lower CFS scores (1.7±0.5, 4.7±1.4, 3.8±1.9, P=0.006) and tear LDH levels (0.10±0.01, 0.19±0.01, 0.16±0.08 OD, P=0.014) but higher tear MUC5AC levels (12.9±3.7, 7.9±2.0, 9.7±3.6 ng/mL, P=0.041) compared with the control and saline-treated groups. There were no significant differences between the control and saline-treated groups. In lacrimal gland-excised rats, the natural extract-treated group also had lower CFS scores (4.3±1.2, 11.5±2.3, 9.0±1.9, P<0.001, P=0.001) and tear LDH levels (0.30±0.08, 0.48±0.12, 0.39±0.05 OD, P<0.05) but higher tear volume (4.3±0.9, 1.9±0.7, 2.8±1.1 mm, P=0.005, P=0.124) and tear MUC5AC levels (8.2±2.0, 2.9±1.2, 5.4±2.2 ng/mL, P<0.001, P=0.047) compared with the control and saline-treated groups. There were no significant differences in the CFS scores, tear MUC5AC level, and tear LDH level between the normal and natural extract-treated groups. CONCLUSION: The natural extract consisting of bee venom, musk, and deer antlers may have effectiveness in DED treatment by restoring the damaged ocular surface, increasing tear volume, and recovering the tear mucin layer in DED rats.