Background Human parental care relies heavily on the ability to monitor and respond to a child’s affective states. The current study examined pupil diameter as a potential physiological index of mothers’ affective response to infant facial expressions. Methods Pupillary time-series were measured from 86 mothers of young infants in response to an array of photographic infant faces falling into four emotive categories based on valence (positive vs. negative) and arousal (mild vs. strong). Results Pupil dilation was highly sensitive to the valence of facial expressions, being larger for negative vs. positive facial expressions. A separate control experiment with luminance-matched non-face stimuli indicated that the valence effect was specific to facial expressions and cannot be explained by luminance confounds. Pupil response was not sensitive to the arousal level of facial expressions. Conclusions The results show the feasibility of using pupil diameter as a marker of mothers’ affective responses to ecologically valid infant stimuli and point to a particularly prompt maternal response to infant distress cues. Electronic supplementary material The online version of this article (doi:10.1186/s12993-017-0120-9) contains supplementary material, which is available to authorized users.