Abstract: The study of physiological and biochemical responses of leaf reddening during overwintering in broadleaf evergreens has theoretical significance to reveal the coloration mechanism and can also provide practical guide for maintaining the green landscape in northern China. In the present study, we measured the contents of pigments and their distributions, the related indexes of reactive oxygen species (ROS) in five different leaf color phenotypes: Green (GG), Green Brown (GB), Brown (BB), Red Brown (RB) and Red (RR) which were subjected to different illumination in Buxus microphylla ‘Belvedere’. The results showed that chlorophylls in green leaves without red region were distributed uniformly across the leaf, while in the other phenotypes, red pigments (when presented) were mostly located in the upper palisade mesophyll and the lower spongy tissue whose red depth was consistent with the phenotype color and the carotenoids accumulation. Compared with GG, winter reddening leaves (GB, BB, RB and RR) suffered from more serious photoinhibition and had higher content of carotenoids (Car). The related indexes of ROS, such as hydrogen peroxide (H2O2) level, and the activity of superoxide dismutase (SOD) and catalase (CAT) also increased significantly. Change of Car content was positively correlated with H2O2 level, and the redder phenotypes had the higher contents in both of the two indexes and the lower photosystems II (PSII) photochemical efficiency (Fv/Fm). The content of total chlorophyll Chl (a+b) was the lowest in RR while the highest in RB. There was no certain pattern for the changes of POD activities in different leaf color phenotypes. Additionally, the contents of superoxide radical (O-2) and malondialdehyde (MDA) in the two reddest phenotypes RB and RR were the lowest and O-2 showed the significantly negative correlation with carotenoids. In summary, these results suggest that the carotenoids accumulation not only has a relationship with the winter leaves reddening but also has a significant correlation with H2O2. The increase of H2O2 might enhance the antioxidant enzyme activity and the upregulation of Car content, which protects the red leaves from oxidative damage. The overwintering leaf color of B. microphylla ‘Belvedere’ is closely related with the ROS, but different ROS types played varying roles in the coloration. H2O2 may play a positive role and it is suggested for further study.