In this study, with an aim to understand the improvement effect and regulatory mechanism of Pseudostellaria heterophylla(Miq.) Pax. polysaccharides on atopic dermatitis mice, the NO levels regulated by P. heterophylla polysaccharides was detected using a macrophage RAW264.7 model induced by LPS, and the atopic dermatitis model in mice was constructed by 2,4-dinitrofluorobenzene and the skin lesions, pathological changes were collected and the content of filaggrin(FLG), claudin-1(CLDN1), interleukin-4(IL-4), interleukin-13(IL-13) and interferon-γ(INF-γ) in skin tissue was measured using ELISA method. According to the results, P. heterophylla polysaccharide prepared in this study could significantly inhibit NO secretion in RAW264.7 cells induced by LPS. Under external treatment with P. heterophylla polysaccharide, the DNFB-induced atopic dermatitis skin symptoms of mice was alleviated, both the rate of transdermal water loss and the thickness of the epidermal layer were reduced, meanwhile, the contents of FLG and CLDN1 proteins were increased and the inflammatory factors IL-4, IL-13 and INF-γ were inhibited. It could be concluded that P. heterophylla polysaccharides could improve the skin barrier and reduce the secretion of inflammatory cytokines, playing a significant role in repairing atopic dermatitis in mice.