Objective To examine the effects of tofacitinib on T follicular regulatory cell / T follicular helper cell (Tfr/Tfh) balance and expression of CXCL13 and transforming growth factor-β1 (TGF-β1) in experimental autoimmune encephalomyelitis (EAE) rats.Methods Fifty female Wistar rats were randomly assigned to the normal control group, EAE control group, and low-dose, median-dose, and high-dose tofacitinib groups (10 rats per group). The rats were given subcutaneous injection of the myelin basic protein of the guinea pig spinal cord and complete Freund’s adjuvant to induce EAE. Rats in the control group and low-dose, median-dose, and high-dose tofacitinib groups were gavaged with normal saline and 1, 2, and 4 mg/kg/d tofacitinib, respectively, for 10 days starting on day 3 before EAE induction. Disease condition, the percentage of splenic Tfh cells and Tfr cells, the change in splenic Tfr/Tfh ratio, and the changes in CXCL13 and TGF-β1 concentrations in brain tissue homogenates were measured and recorded.Results The incubation period was significantly prolonged in the low-dose (16.70±1.50 d), medium-dose (20.20±2.44 d), and high-dose (22.90±1.79 d) tofacitinib groups than in the EAE control group (10.20±1.99 d) (all P<0.01). In contrast, the progressive stage was significantly shortened in the low-dose (8.00±2.00 d), medium-dose (5.60±1.51 d), and high-dose (3.00±1.16 d) tofacitinib groups than in the EAE control group (10.50±1.84 d) (all P<0.01). Furthermore, the neurological dysfunction score was significantly reduced in the low-dose (2.30±1.34 points), medium-dose (1.20±1.40 points), and high-dose (0.60±0.84 points) tofacitinib group than in the EAE control group (3.80±1.03 points) in the peak period of disease onset (all P<0.01). Compared with the EAE control group, the tofacitinib groups had significantly reduced percentage of Tfh cells and CXCL13 concentration and significantly increased percentage of Tfr cells, Tfr/Tfh ratio, and TGF-β1 concentration, all in a dose-dependent manner (P<0.01; P<0.05).Conclusions Tofacitinib has a dose-dependent preventive and therapeutic effect on EAE in rats. Its preventive and therapeutic effect may be associated with the downregulation of the percentage of Tfh cells and expression of CXCL13, upregulation of the percentage of Tfr cells and expression of TGF-1, and the increase in Tfr/Tfh ratio.