To analyze the expression of toll-like receptor 9 mRNA, TNF (tumor necrosis factor)-α and IL (interleukin)-6 after the stimulation of murine microglia (BV2) by hepatitis C virus (HCV) so as to elucidate the immune pathogenesis of HCV in the injury of central nervous system (CNS). BV2 cells were cultured in vitro and divided into 3 groups: HCV-positive serum group, normal serum group (negative control) and blank control group. Each group had 20 samples. The cells and supernatant were collected at 4, 12, 20, 36 h. And TLR9 mRNA was detected by RT-PCR (reverse transcription-polymerase chain reaction) and the levels of TNF-α and IL-6 were detected by ELISA (enzyme-linked immunosorbent assay). Comparison analyses were performed by SPSS13.0 statistical package. (1) RT-PCR showed that there was the expression of TLR9 mRNA in BV2 cells. And the level of TLR9 mRNA in HCV-positive serum group (0.85 ± 0.11, 0.55 ± 0.08, 0.58 ± 0.09, 0.61 ± 0.07) was higher than that in the other groups. The difference had statistical significance (F = 17.258, P < 0.05). It was the highest at 4 h (0.85 ± 0.11). And the difference had statistical significance (F = 7.427, P < 0.05); but there were no statistical significance between normal serum group and control group (P > 0.05); no significant difference existed among 12, 20 and 36 h (P > 0.05). (2) The levels of TNF-α [(926 ± 133), (327 ± 59), (367 ± 69), (354 ± 71) pg/ml] and IL-6 [(125 ± 34), (84 ± 20), (82 ± 24), (87 ± 18) pg/ml] in the HCV-positive serum group were higher than those in the other groups (F = 18.263 - 27.469, P < 0.05); in HCV-positive serum group, the levels of TLR9 mRNA and TNF-α (r = 0.425, P < 0.01)/IL-6 (r = 0.489, P < 0.01) were positively correlated. The expression of TLR9 mRNA is elevated after the stimulation of BV2 cells by HCV-positive serum. It peak comes at 4 h. And it may induce the secretions of TNF-α and IL-6. Thus TLR9 may increase the secretions of Th1 and Th2 cytokines so as to participate in the early inherent immune response during the infection of CNS by HCV.