OBJECTIVE To investigate the effect of a new non-selective muscarinic antagonist, 0.05% racanisodamine eye drop, on pupil size and accommodative response in children. METHODS Twenty healthy myopic children aged between 9 and 12 years were enrolled in the study. They were given two successive drops of 0.05% racanisodamine solution in one eye. Scotopic pupil sizes of both eyes were evaluated with an infrared open-field autorefractor before and 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, and 360 min after treatment. Accommodative responses to a Maltese cross at a distance of 50, 33, and 20 cm were measured at the same time intervals as pupil sizes. Subjective evaluation of photophobia and reading difficulty were recorded by questionnaires. Pupil sizes and accommodative responses were compared across time using repeated measurements of analysis of variance. RESULTS The minimum pupil size of the treated eye came at 10 min after the second drop and maintained for the next 20 min. Then, it recovered to baseline value at 45 min and continued to enlarge to a peak size of about 0.75 ± 0.22 mm larger than baseline value at 120 min (p = 0.036), followed by a recovering process. No significant change occurred to the contralateral untreated eye. Accommodative responses to near targets did not significantly change at any time during the procedure for both eyes. Only one subject experienced photophobia in the treated eye at the 120 and 180 min time points after treatment. None reported reading difficulty at any time. CONCLUSIONS As a non-selective muscarinic antagonist, 0.05% racanisodamine has a significant but clinically moderate impact on pupil size and no effect on accommodative response.