This study aims to assess the effect of topical 0.5% apraclonidine on Intraocular pressure (IOP) in horses and compare the effects of timolol maleate 0.5% with 0.5% apraclonidine in the equine eye. Twenty healthy female thoroughbred horses were used. Horses were divided into two groups. Ten horses received single dose of 0.2 mL of 0.5% apraclonidine in one randomly selected eye and the contralateral eye received single dose of 0.2 mL of artificial tears. In the second group, 10 horses received single dose of 0.2 mL of 0.5% timolol maleate in one eye and the opposite eye received single dose of placebo (0.2 mL of artificial tears). Intraocular pressure was measured using rebound tonometer at the baseline and 30, 60, 120, 240, 360 minutes, and 24 hours after topical ophthalmic drops instillation. Any ocular side effects were recorded at each time point. Mean (SD) baseline IOPs of the treated and placebo eyes were 26.2(3.1) and 23.5(3.4) in apraclonidine group, and 25.7(2.6) and 23.2(3.3) in timolol group. In the apraclonidine group, significant reduction in the mean IOP started after 60 minutes (P= .005) and was still present after 24 hours (P < .001). In timolol group, IOP was reduced in the treated eyes, but this reduction was only significant in the treated eyes at T24h (P= .03). The highest reduction in IOP in timolol group was observed at T360 (21.0(2.2); 14.7%). Mean IOP was decreased prominently by apraclonidine compared to timolol in treated eyes. In conclusion, single dose of topical 0.5% apraclonidine reduced IOP significantly among normal horses in the present study. Further investigations are necessary for evaluating efficacy and safety of apraclonidine in horses.