Exosomes, especially tumor-derived exosomes (TEXs), are involved in a wide kinds of biological activities. However, the mechanism of interaction between TEXs and immune cells, such as natural killer (NK) cells, still remains unclear. Here, we isolated the TEXs secreted from oral cancer (OC) cells by ultrafiltration and affinity chromatography methods and examined the effect of the TEXs on NK cells. The TEXs could be internalized by NK cells and then promoted the biological functions of NK cells, such as proliferation, cytotoxicity, release of perforin and granzyme M. Overexpression of the Interferon Regulatory Factor 3 (IRF-3) protein was determined in NK cells after treated with TEXs. Furthermore, the NF-κB-Activating Kinase Associated Protein 1 (NAP1), the upstream activator of IRF-3, was found enriched in TEXs. Up-regulated of NAP1, IRF-3 and p-IRF-3 were determined in NK cells treated with TEXs. In addition, as the downstream molecules of IRF-3, the type I interferon (IFNα) gene, the chemokine (C-X-C motif) ligands (CXCL) gene and other genes, were also been transcriptionally activated. In contrast, the NAP1-depleted TEXs obtained from OCs dramatically weakened the interaction between the TEXs and NK cells. Hence, our study suggests that the exosomal NAP1 derived from oral cancer cells could enhance the cytotoxicity of NK cells. This work was supported by the National Program on Key Research Project of China (2016YFC0902700) and the National Natural Science Foundation of China (81472572 and 91229103).