Variations in pupil size under constant luminance are closely coupled to changes in arousal state [1][2][3][4][5]. It is assumed that such fluctuations are primarily controlled by the noradrenergic system [6][7][8][9]. Phasic activity of noradrenergic axons precedes pupil dilations associated with rapid changes in arousal [7,9], and is believed to be driven by unexpected uncertainty [1,[10][11][12][13][14][15][16]. However, the role of other modulatory pathways in the control of pupil-linked arousal has not been as thoroughly investigated, but evidence suggests that noradrenaline may not be alone [7,17,18]. Administration of serotonergic drugs seems to affect pupil size [19][20][21][22][23], but these effects have not been investigated in detail. Here, we show that transient serotonin (5-HT) activation, like noradrenaline, causes pupil-size changes. We used phasic optogenetic activation of 5-HT neurons in the dorsal raphe nucleus (DRN) in head-fixed mice locomoting in a foraging task. 5-HT-driven modulations of pupil size were maintained throughout the photostimulation period and sustained for several seconds after the end of the stimulation. The activation of 5-HT neurons increased pupil size additively with locomotor speed, suggesting that 5-HT transients affect pupil-linked arousal independently from locomotor states. We found that the effect of 5-HT on pupil size depended on the level of environmental uncertainty, consistent with the idea that 5-HT may report a salience or surprise signal [24] . Together, these results challenge the classic view of the neuromodulatory control of pupil-linked arousal, revealing a tight relationship between the activation of 5-HT neurons and changes in pupil size.