Interleukin (IL)-35 is a novel anti-inflammatory cytokine that is produced by regulatory T cells. IL-35 is reported to suppress IL-17A-producing helper T (Th17) cell activation. IL-17A is related to progression of periodontitis. Furthermore, IL-35 and IL-17A are detected in human gingival crevicular fluid. However, the effect of IL-35 and interaction between IL-35 and IL-17A on pro-inflammatory cytokine production in human periodontal resident cells are still unclear. The aim of this study was to clarify the effect of IL-35 on IL-6 and IL-8 production in human periodontal ligament cells (HPDLCs) stimulated with IL-17A. IL-35 inhibited IL-6 and IL-8 production in IL-17A-stimulated HPDLCs. Moreover, western blot analysis showed that IL-35 suppressed extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB p65 phosphorylation in IL-17A-stimulated HPDLCs. Our findings suggested that IL-35 produced from regulatory T cells might inhibit progression of periodontitis by decreasing IL-17A-induced levels of IL-6 and IL-8.