In our article, we evaluated the regulatory effects of the infusions of rituximab, a monoclonal antibody directed against CD20(+) B cells, on the serum matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases-1 (TIMP-1) levels in patients with active rheumatoid arthritis (RA) not responding to anti-tumor necrosis factor (anti-TNF) therapy. Twelve RA patients were planned to receive four infusions of 1,000 mg of rituximab at weeks 0, 2, 24 and 26. The therapy was combined with methotrexate (MTX) (20-30 mg/week). Seven patients were refractory to previously received infliximab, and five to etanercept. Serum concentrations of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase B (MMP-9) and TIMP-1 were measured by ELISA on weeks 0, 2, 12, 24, 36 and 52. Initial infusion of rituximab downregulated serum MMP-1 (p < 0.01), MMP-3 (p < 0.001), MMP-9 (p < 0.001) and TIMP-1 (p < 0.05) levels. Second drug administration caused even more remarkable reduction of measured MMPs (p < 0.001 in all cases) and TIMP-1 level (p < 0.01). These findings were accompanied by significantly decreased ratios of measured MMPs to TIMP-1. Next rituximab infusions on weeks 24 and 26 sustained the suppression of serum MMPs levels. Prior to the initial rituximab infusion, serum concentrations of studied MMPs and TIMP-1 significantly correlated with markers of RA activity such as disease activity score (DAS28) and CRP levels. Rituximab therapy, beside a rapid clinical improvement, reduced serum MMPs concentrations in RA patients refractory to anti-TNF treatment. Repeated infusions of rituximab maintained initial serum MMPs suppression.