〔Abstract〕 Objective To investigate the effect of organic anion transporter1(OAT1) on the absorption of methotrexate(MTX) and its proliferation and migration in fibroblast-like synovial cells(FLS) of rats with collagen induced arthritis(CIA). Methods The CIA model was established in rats and the FLS was isolated and cultured. The transfection technique was used to up-regulate the expression of OAT1 in FLS, and Western blot and RT-qPCR were used to verify the transfection efficiency. FLS were divided into negative control(NC) and OAT1 overexpression groups and stimulated with tumor necrosis factor-α(TNF-α)(20 μg/L). In addition, normal rats FLS were taken as the Control group. The MTX and p-aminohippuric acid(the specific substrate of OAT1, PAH) were given at the same concentration( 100 ng/ml). And the absorption function of FLS for MTX and PAH was evaluated by UPLC-MS/MS/MS. At the same time,after MTX( 100 ng/ml) was administered,the proliferation effect and migration ability of FLS were detected by CCK-8 and Transwell. Results Compared with the NC group,the expressions of OAT1 and Slc22 a6 in the overexpression group significantly increased. Compared with the NC group,the absorption of MTX and PAH by FLS in the OAT1 overexpression group significantly increased at 15,30,45,60 and 120 min. Compared with the NC group,the proliferation of FLS in the OAT1 overexpression group significantly decreased at 12 h and approached the level of the Control group. Compared with the NC group,the migration ability of OAT1 overexpression group decreased significantly after 12 h and approached the level of the Control group. Conclusion The increased expression of OAT1 can significantly increase the absorption of MTX by inflammatory FLS and inhibit its abnormal proliferation effect and migration ability.