MicroRNAs (miRNAs) are demonstrated to contribute to the regulation of drug resistance in a number of diseases. Nevertheless, little is known about the role and the underlying mechanism of miR-16 in rheumatoid arthritis (RA) methotrexate resistance. In this study, we firstly examined the miR-16 expression in the serum and synovial fluid from RA patients who were unresponsive to methotrexate monotherapy (UR-MTX patients) and responsive RA patients (R-MTX patients). Secondly, the miR-16 expression was measured in both fibroblast-like synovial cells (FLS) and methotrexate resistance RA-FLS cells (FLS-MTX). FLS cells used in this study were isolated from synovial tissue specimens obtained from patients with RA who underwent total joint replacement. FLS-MTX cells were conducted by gradually increasing the concentration of methotrexate in the medium. The construction of FLS-MTX cells was confirmed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. Thirdly, in order to further investigate the role of miR-16 in FLS-MTX cells, we introduced miR-16 inhibitor into FLS-MTX cells to knockdown the expression of miR-16, used fluorescence quantitative PCR to detect the inhibition efficiency. The effects of miR-16 inhibition on cell viability, cell cycle arrest and apoptosis in FLS-MTX cells were monitored with MTT and flow cytometry analysis, respectively. And the regulation of miR-16 on P-glycoprotein (P-gp) was performed using qRT-PCR, western blotting, and immunofluorescence staining. Fourthly, ammonium pyrrolidinedithiocarbamate (PDTC), a NF-κB pathway inhibitor, was applied to verify the mechanism by which miR-16 involved in to regulate the P-gp expression, and thus contributing to the methotrexate resistance in FLS-MTX cells. MiR-16 was upregulated in the in serum and synovial fluid from UR-MTX patients as well as in FLS-MTX cells. Inhibition of miR-16 re-sensitized the FLS-MTX cells to methotrexate by suppressing the cell viability, cell promoting cycle arrest at G0/G1 phase and enhancing apoptosis. Knockdown of miR-16 significantly reduced MDR1 mRNA expression and P-gp protein expression in FLS-MTX cells. Furthermore, inhibition of NF-κB pathway by PDTC reinforced the effect of miR-16 knockdown on P-gp expression, cell viability, cell cycle arrest and apoptosis. In conclusion, our study illustrated that inhibition of miR-16 in FLS-MTX cells alleviated methotrexate resistance by inhibiting MDR1/P-gp expression through inactivation of the NF-κB pathway.