Aims: The roles of specific active ingredients in Chinese medicinal formulas have not been clearly elucidated. In this study, we selectively deleted and replenished paeoniflorin from Si-Ni-San, a traditional Chinese prescription, and aimed to identify the molecular basis of how paeoniflorin exerted its effect in Si-Ni-San. Main methods: Contact dermatitis was induced in mice with picryl chloride. Paeoniflorin was selectively deleted from Si-Ni-San by an immunoaffinity column. Quantitative real-time PCR, western blot, and enzymelinked immunosorbent assay were used in this study. Key findings: Both Si-Ni-San and paeoniflorin significantly reduced ear swelling in mice while the paeoniflorindeleted Si-Ni-San (Si-Ni-SanPF− ) showed little ameliorative effect. In lipopolysaccharide-evoked macrophages, SiNi-San and paeoniflorin markedly inhibited tumor necrosis factor-α production, cyclooxygenase-2 activity, as well as extracellular signal-regulated kinase 1/2 phosphorylation while Si-Ni-SanPF− exhibited no or slight inhibitory effect. Furthermore, the inhibitory effect on the production of tumor necrosis factor-α reappeared when different proportions of paeoniflorinwere replenished in Si-Ni-SanPF− . In addition, the expression of macrophage migration inhibitory factor in T cells, rather than macrophages, was significantly inhibited by Si-Ni-San, but not Si-Ni-SanPF−. Our data indicate paeoniflorinis the principal component of Si-Ni-San, exerting negative regulation on the function of macrophages in contact dermatitis.