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-evokedmacrophages, Si-Ni-San and paeoniflorinmarkedly inhibited tumor necrosis factor-αproduction, cyclooxygenase-2 activity, aswell 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 ofmacrophagemigration inhibitory factor in T cells, rather thanmacrophages,was significantly inhibited by Si-Ni-San, but not Si-Ni-SanPF-.Ourdata indicate paeoniflorin is the principal component of Si-Ni-San, exertingnegative regulationon the function of macrophages in contact dermatitis. Significance: The present study suggests that dissecting the role of specific constituents in medicinal formulas through selective deletion and replenishment may be a useful strategy in recognizing and validating an active ingredient in traditional Chinese medicine.