Studies indicate that the natural immune-related protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) plays an antitumor role in nasopharyngeal epithelial tissue. However, the detailed mechanism of the tumor-suppressor effect of SPLUNC1 in the inflammatory microenvironment of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) remains elusive. The aim of the present study was to explore how SPLUNC1 reduces the inflammatory response of NPC cells infected with EBV by regulating the Toll-like receptor (TLR)9/NF-κB signaling pathway. As detected by immunohistochemistry and western blotting, SPLUNC1 protein expression exhibited low or negative expression in the NPC epithelial samples/cells, while it demonstrated positive expression in normal nasopharyngeal epithelial tissues/cells; this pattern of expression was the contrary to that of TLR9. The poorly differentiated HNE2 cell line had the highest efficiency of transfer of infection with EBV by 'cell-to-cell' contact method. The group of EBV-infected HNE2 cells showed significantly higher activation of the expression of TLR9/NF-κB signaling pathway-associated factors (TLR9, CD14, MyD88, IKK, P-IKβα, P-NF-κB and NF-κB). The levels of inflammatory cytokines IL-6, IL-8, IL-1β and TNF-α in the HNE2 cell group after EBV infection were higher than these levels in the uninfected cell group (P<0.05); Meanwhile, after EBV infection, the expression levels of TLR9/NF-κB pathway associated-protein and inflammatory cytokines IL-6, IL-8, IL-1β and TNF-α in the HNE2/SPLUNC1 cell group were lower than these levels in the HNE2/Vector cell group (P<0.05). After EBV-DNA direct transfection, cytokine mRNA expression levels of TLR9, IL-6, IL-8, IL-1β and TNF-α in the HNE2 cell group were significantly higher than these levels in the NP69 cell group (P<0.05). The expression levels of these cytokines in the HNE2/SPLUNC1 cell group were obviously lower than these levels in the HNE2/Vector cell group (P<0.05). These results suggest that EBV infection of NPC cells can activate the TLR9/NF-κB signaling pathway, promote the release of inflammatory cytokines and consequently enhance the inflammatory response, while SPLUNC1 can weaken the inflammatory response induced by EBV infection in NPC cells through the regulation of the TLR9/NF-κB signaling pathway and control of the tumor inflammatory microenvironment.