Toll-like receptor 9 (TLR9) plays a pivotal role in sensing a wide range of pathogens, including bacteria, fungi, and viruses. A dysregulation of TLR9 signaling may contribute to a higher risk of developing cancers. A hospital-based case-control study, including 356 nasopharyngeal carcinoma (NPC) cases and 356 controls, was conducted to assess the relationship between TLR9 -1237T/C, -1486T/C, and 2848G/A polymorphisms and NPC risk as well as clinical characteristics. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Protein level of transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and interleukin-6 (IL-6) in NPC biopsies was measured by enzyme-linked immunosorbent assay (ELISA). We found that -1486T/C CC genotype had an increased NPC risk at odds ratio (OR) = 1.808 with 95% confidence interval (CI) 1.169 ∼ 2.798 (P = 0.008). The patients with -1486 CC genotype are inclined to advanced tumor stage and lymph node metastasis. In addition, protein concentration of VEGF in NPC biopsies with -1486 CC genotype was significantly increased compared patients with -1486 TT genotype. For the first time, our data suggested that TLR9 -1486T/C may be a risk biomarker of NPC.