The reward system may provide an interesting intermediate phenotype for anhedonia in affective disorders. Reward anticipation is characterized by an increase in arousal, and previous studies have linked the anterior cingulate cortex (ACC) to arousal responses such as dilation of the pupil. Here, we examined pupil dynamics during a reward anticipation task in forty-six healthy human subjects and evaluated its neural correlates using functional magnetic resonance imaging (fMRI). Pupil size showed a strong increase during monetary reward anticipation, a moderate increase during verbal reward anticipation and a decrease during control trials. For fMRI analyses, average pupil size and pupil change were computed in 1-s time bins during the anticipation phase. Activity in the ventral striatum was inversely related to the pupil size time course, indicating an early onset of activation and a role in reward prediction processing. Pupil dilations were linked to increased activity in the salience network (dorsal ACC and bilateral insula), which likely triggers an increase in arousal to enhance task performance. Finally, increased pupil size preceding the required motor response was associated with activity in the ventral attention network. In sum, pupillometry provides an effective tool for disentangling different phases of reward anticipation, with relevance for affective symptomatology.