To investigate the correlation between TLR9 and cytokine secretion in SLE diagnosis and treatment. A total of 66 cases (39 SLE and 27 healthy donors) were enrolled in this study. The CD20+ labeled B cells were isolated from SLE patients. TLR9 mRNA expression from SLE tissues and B cells was detected using RT-PCR. The cytokine secretion in B cells were measured using ELISA. Correlation between TLR9 expression and cytokines secretion was analyzed using gene silencing method. Compared with the controls, TLR9 expression was significantly high in SLE patients tissues, as well as in B cells. Expressions of IL-6 and ds-DNA antibody were high in SLE patients serum and were positively correlated with TLR9 level in SLE patients (IL-6, R(2) = 0.768; ds-DNA antibody, R(2) = 0.730). The IL-6 and ds-DNA expression were significantly decreased by silencing TLR9 compared to the controls. Moreover, silencing TLR9 significantly decreased cytokines secretion including IL-6, IL-10, and IL-1rα, as well as the pathway-associated protein expression, including ICOS and Foxp3. The successful application of TLR9 silencing method in human SLE B cells may loan theatrical basis for the possibility of TLR9 genetic therapy in SLE diagnosis and treatment.