Pseudostellaria heterophylla (PH) has various pharmacological effects that include immunologic enhancement and anti-oxidation. However, it remains unclear whether PH exerts beneficial effects in dermatological diseases. The present study examined the effects of PH on a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD) mouse model and elucidated its underlying mechanism of action. PH extract (1 and 100 mg/ml) was applied topically to DNCB-treated dorsal skin of mice every day for 11 days. The immunomodulatory effects of PH were evaluated by measuring skin thickness, mast cell infiltration, serum levels of immunoglobulin E (IgE), and mRNA expression levels of T helper (h)1/Th2 and pro-inflammatory cytokines in dorsal skin. In addition, cluster of differentiation (CD)4(+) T cells were detected in dorsal skin by immunohistochemistry. Topical application of PH significantly reduced the thickness of dermis, epidermis and serum IgE production compared with the DNCB group. PH treatment inhibited infiltration of inflammatory cells, including mast cells and CD4(+) T cells, and suppressed the mRNA expression levels of cytokines (interferon-gamma, interleukin-4, -6, -8 and -1 beta, and tumor necrosis factor-alpha) associated with the immune response. Furthermore, PH treatment significantly downregulated the protein expression levels of nuclear factor-kappa B, phosphorylated inhibitor of kappa B alpha and mitogen-activated protein kinases. The results suggested that PH may be a potential therapeutic strategy for the treatment of AD via the modulation of Th1 and Th2 levels.