This study explores the mechanisms and potential targets of paeoniflorin (Pae) in treating atopic dermatitis (AD) through network pharmacology, molecular docking, and experimental validation. Pae, an active ingredient from Paeonia lactiflora Pall., exhibits immunomodulatory and anti-inflammatory effects. Literature from PubMed identified Pae, and its potential targets were predicted using tools like Swiss Target Prediction, Target Net, Super-PRED and Pharm Mapper databases. AD-related targets were obtained from DisGeNET and Gene Cards, and their intersection was analyzed using Venny. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified key pathways. Molecular docking confirmed Pae's interaction with core targets. In vivo, AD was induced in mice using calcipotriol (MC903), and Pae's effects were assessed through dermatitis score, epidermal thickness, spleen index, and histopathology. In vitro, TNF-α-induced Human immortalized keratinocyte (HaCaT) cells were used to assess inflammation and apoptosis. Enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analyzed inflammatory markers, while Hoechst staining measured apoptosis. Network pharmacology identified 229 predicted targets for the ingredient Pae, and 1458 disease-associated targets for AD, finally revealed 63 common genes between them. Protein-Protein Interaction (PPI) analysis revealed 17 core targets. Experimental results showed Pae reduced dermatitis scores, epidermal thickness, spleen index, and inflammatory factors (IL-1β, IL-6, IgE, TNF-α, IL-8). Hoechst staining showed reduced apoptosis, and WB analysis revealed Pae's effects on MAPK and Apoptosis signaling pathways. These findings suggest Pae's potential for AD treatment.