Abstract Background Intraocular pressure (IOP) may vary according to the change of ocular conditions. In this study, we want to assess the effect and mechanism of pupil dilation on IOP in normal subjects. Methods We prospectively evaluated 32 eyes of 32 patients (age; 61.7 ± 8.2 years) with normal open angles under diurnal IOP. IOP was measured every two hours from 9 AM to 11 PM for one day to establish baseline values and was measured again for one day to assess the differences after dilation. To induce dilation, we administered 2.5% phenylephrine and 1% tropicamide every 5 minutes from 8:30 AM to 8:45 AM and for every two hours from 11 AM to 9 PM to keep the pupil dilated. Diurnal IOP, biometry, Visante OCT, and laser flare photometry were measured before and after dilation. Results We observed a significant increase in IOP after dilation, 1.85 ± 2.01 mmHg (p = 0.002). IOP elevation remained significant until about four hours after dilation. Thereafter, IOP decreased slowly and eventually reached pre-dilation level (p > 0.05). Flare values decreased, and the anterior chamber angle became wider after mydriasis. Conclusions Dilation of the pupil significantly and incidentally elevated IOP in normal subjects. Further related studies are warranted to characterize the mechanism of the increased IOP after dilation.