Pseudolaric acid B (PAB) is a novel diterpenoid, isolated from Pseudolarix kaempferi Gorden, which roots are widely used to treat inflammatory and microbial skin diseases for centuries, but the underlying mechanism remains elusive. To address the immunoregulatory mechanisms of PAB, we first investigated the effect of PAB in 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS) mice. The expression of specific transcription factors for T-helper (Th)1, Th2, Th17 and regulatory T cells (Tregs) was determined by RT-PCR, and the capacities for PAB to modulate Th1/Th17/Treg cell subsets was furtherly analyzed by flow cytometric analysis, ELISA and Western blotting assay. The results showed that topical application of PAB could suppress ear swelling, block inflammatory infiltration, and interfere in Th1 response. Furthermore, PAB-treated CHS mice exhibited the impaired Th17 development and enhanced Tregs generation, associated with the up-regulation of peroxisome proliferator activated-receptor γ (PPAR γ) expression. These findings suggest that PAB exerts its immunoregulatory activity through regulating the balance of Th1/Th17/Treg cell subsets, which would provide a novel therapeutic application for PAB on CHS and other inflammatory-associated diseases.