SUMMARY Pupil size is collectively controlled by the sympathetic dilator and parasympathetic sphincter muscles. Locus coeruleus (LC) activation has been shown to evoke pupil dilation, yet how the sympathetic and parasympathetic pathways contribute to this dilation remains unknown. We examined pupil dilation elicited by LC activation in lightly anesthetized rats. Unilateral LC activation evoked bilateral, yet lateralized pupil dilation, i.e, the ipsilateral dilation was significantly larger than the contralateral dilation. Surgically blocking the ipsilateral, but not contralateral, sympathetic pathway significantly reduced lateralization, suggesting lateralization is mainly due to sympathetic contribution. Moreover, we found that sympathetic, but not parasympathetic, contribution is correlated with LC activation frequency. Together, our results unveiled the frequency-dependent contributions of the sympathetic and parasympathetic pathways to LC-activation-evoked pupil dilation, and suggest that lateralization in task-evoked pupil dilations may be used as a biomarker for autonomic tone.