Findings regarding the direction of pupil modulation (dilation/constriction) related to aesthetic preference are often contradictory, partly because preference is determined by multiple factors depending on the target. Research has demonstrated that art paintings with image features similar to those of natural scenes are preferred, suggesting that the perception of naturalness is one factor affecting preference. This study aimed to test the hypothesis that the pupil dilation in response to visual examination of a preferred painting may be caused by the perceived naturalness of the color composition of the painting. We investigated the relationship between pupil response and preference/perception of naturalness by manipulating the color composition of art paintings. First, we found that when compared to hue-rotated paintings, the paintings presented with their original color composition were preferred and perceived as more natural. Second, the extent of the pupil dilation had a significant positive correlation with the perceptions of naturalness for the painting, whereas there was no significant correlation between pupil dilation and preference. This result indicates that the pupil dilation during the viewing of the paintings is not caused by preference but by the perceived naturalness regarding the color composition of paintings. This was observed not only in representational paintings but also in abstract paintings, suggesting that the color composition of a painting itself, rather than memory colors-the standard, known hues of objects-or other influences, is an important factor of perceived naturalness that produces pupil dilation.