Toll-like receptors (TLRs) play a crucial role in the innate immune response to pathogens, and TLR3 could recognize and control the herpesvirus. We studied the effect of TLR3 polymorphisms on the risk of Kaposi's sarcoma-associated herpesvirus (KSHV) infection. A cross-sectional study was performed among human immunodeficiency virus (HIV)-infected individuals in Xinjiang, a KSHV-endemic region of China. The frequencies of nine single-nucleotide polymorphisms (SNPs) in TLR3 in 370 KSHV-infected patients and 558 controls, and their impact on plasma IFN-γ levels, were compared. The effect of TLR3 SNPs on the KSHV viral load in KSHV-infected subjects was also assessed. The minor allelic variant at rs13126816 was more common among KSHV-seronegative than KSHV-infected individuals. Two TLR3 SNPs (rs13126816 and rs3775291) showed a protective effect against KSHV infection (rs13126816: odds ratio [OR]dominant = 0.66, 95% confidence interval [CI]: 0.50-0.87; ORoverdominant = 0.65, 95% CI: 0.49-0.87; rs3775291: ORdominant = 0.76, 95% CI: 0.58-0.99; ORoverdominant = 0.75, 95% CI: 0.57-0.98). These associations were stronger in the Uyghur compared with the Han population. The haplotype, CGAC, significantly correlated with the risk of KSHV infection (OR = 0.72, p = 0.029). KSHV-infected individuals with homozygous rs13126816 AA genotypes had a lower KSHV viral load (aOR = 0.14; p = 0.038). However, no association was observed between TLR3 SNPs and plasma levels of IFN-γ. Genetic variants in TLR3 reduce the risk of KSHV infection and affect KSHV reactivation among HIV-infected individuals, especially in the Uyghur population.