Objective To investigate the therapeutic effect of leflunomide on mesangial proliferative glomerulonephritis and the role of TLR4 signaling pathway in rat mesangial proliferative glomerulonephritis. Methods A total of 36 normal male Sprague-Dawley rats were randomly divided into 3 groups: normal control group (n=12), model group (n=12) and treatment group (n=12). Rats in each group were tested 24-hour urinary protein excretion, serum cystatin C and serum creatinine levels in the end of 4,8 and 12 weeks to observe the mesangial proliferative extent of renal tissue under light microscope after HE staining . Two-step immunohistochemical methods were used to detect the changes in the expression of TLR4, NF-κB, cytokines IL-6 and TNF-α. Results Levels of 24-hour urinary protein excretion, serum cystatin C, serum creatinine, mesangial proliferation of renal tissue, TLR4, NF-κB cytokine IL-6 and TNF-α in model group and treatment group were higher than those in normal control group (P<0.05),levels in treatment group were significantly lower than those in model group (P<0.05). Conclusions Leflunomide has a therapeutic effect on mesangial proliferative glomerulonephritis by inhibiting TLR4 signaling pathway and NF-κB cytokine IL-6 and TNF-α.