Chronic hepatitis C virus (HCV) infection is a worldwide threat to public health. Toll‐like receptor 8 (TLR8) is critical for eliminating RNA viruses, and variation within the TLR8 gene may alter the function of TLR8 in response to HCV infection. Our previous study demonstrated that the TLR8‐129G>C (rs3764879) and TLR8+1G>A (rs3764880) variants were in complete linkage disequilibrium, and that the frequency of TLR8‐129C/+1A was significantly higher in male patients with HCV infection compared with the healthy controls. In the present study, we found that the promoter activity of TLR8‐129G was higher than that of TLR8‐129C in THP‐1 cells. Moreover, TLR8‐129G mRNA stability and competitive DNA‐binding ability were significantly lower than that of TLR8‐129C. To investigate the functional effects of TLR8 polymorphisms, we compared the nuclear factor‐κB (NF‐κB)‐driven luciferase activity in HEK293 cells transfected with the TLR8 variants. TLR8+1A plasmids induced less NF‐κB signalling than did those transfected with TLR8+1G after 20 μm CL075 (P = 0·011) stimulation. We also analysed the mRNA expression and cytokine production in whole blood and monocytes from people of various genotypes stimulated ex vivo by the interferon‐γ and TLR7/8 agonist CL075, R848. TLR8 expression in CD14⁺ cells derived from volunteers with TLR8‐129G/+1G was significantly higher than that derived from TLR8‐129C/+1A, and interleukin‐12p40 production was higher in volunteers with TLR8‐129G/+1G after stimulation. The data indicate that variations in TLR8 genes may modulate immune responses during HCV infection.