Background: Melanoma is a common and malignant cutaneous tumor, which is responsible for a large proportion of skin cancer deaths. Dendritic cell (DC)-based vaccines have achieved positive results in the treatment of melanoma because of their ability to induce cytotoxic response to facilitate tumor elimination. Objective: To improve the efficacy of dendritic cell-based vaccines by the adjuvant activity of Helicobacter pylori neutrophil activating protein (HP-NAP), which is a virulence factor of Helicobacter pylori, has been proved as a TLR agonist with effective immunomodulatory activity. Methods: The recombinant HP-NAP (rHP-NAP) was expressed by using prokaryotic expression system. Dendritic cells (DCs) were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. After treating with rHP-NAP, the maturation of DCs and dendritic cell-based vaccine were assayed by using flow cytometry and qRT-PCR. The activation and proliferation of T cells were measured by FCM, ELISA and MTT methods. The tumor specific cytotoxic response to resistant B16F10 was detected by using lactate dehydrogenase-release assay and qRT-PCR. Results: The recombinant HP-NAP (rHP-NAP), prepared from E. coli prokaryotic expression system, was able to significantly promote the maturation of dendritic cell-based vaccine loaded with tumor cell lysate (TCL) of B16F10 (DC-B16F10-TCL). Furthermore, it effectively induced the activation and proliferation of T cells and tumor specific cytotoxic response to resistant B16F10 melanoma tumor cells. Conclusion: These results suggested that rHP-NAP possesses the potential for use as an adjuvant of dendritic cell-based vaccine in anti-melanoma treatment.