Introduction: Heat shock protein 40 (HSP40) is a vaccine adjuvant candidate for Streptococcus pneumoniae. The mechanism by which HSP40 activates the human dendritic cells (DCs) is unclear.Methods: DCs were isolated from human peripheral blood and their markers (HLA-DR, CD86, CD83, and CD80) were detected by flow cytometry. The messenger RNA (mRNA) and secretion levels of inflammary cytokines were measured after DCs were stimulated with recombinant HSP40 (rHSP40). Short hairpin RNAs were used to knock down toll-like receptor 2 (TLR2) and TLR4. The TLR2-or TLR4-deficient DCs were treated with lipopolysaccharides, rHSP40, or peptidoglycan, and then the secretion levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. Moreover, the secretion levels of TNF-α and IL-6 were measured after DCs were treated with mitogen-activated protein kinase (MAPK) inhibitors including SB203580, SP600125, and U0126. In addition, the phosphorylation levels of p38 MAPK and Jun N-terminal kinase (JNK) in DC cells were determined using western blot analysis after treatment with rHSP40 for different times.Results: DCs were successfully isolated and cultured. rHSP40 treatment significantly increased cytokine levels in a concentration-dependent manner. TLR4 deficiency, but not TLR2 deficiency, significantly suppressed the rHSP40-induced secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). SB203580 and SP600125 significantly inhibited the rHSP40-induced secretion of TNF-α and IL-6. rHSP40 significantly enhanced the phosphorylation of p38 MAPK and JNK.Conclusion: HPS40 stimulates the immune response of DCs via the p38 MAPK and JNK signaling pathways, which depend on TLR4.