Abstract: Objective To investigate the potential role of deoxyribonuclease I (DNase I) in the pathogenesis of rheumatoid arthritis (RA). Methods DNase I activity was measured by radial enzyme-diffusion method in serum samples from 83 RA patients and 60 healthy volunteers and in the synovial fluid (SF) from 27 RA patients and 38 patients with other inflammatory arthritis. SF cfDNA level was measured with Pico Green Kit, and the correlation among DNase I activity, cfDNA level and clinical parameters of RA patients was analyzed. Results Serum DNase I activity was significantly lower in RA patients than in the healthy control subjects (0.3065±0.1436 vs 0.4289±0.1976 U/mL, P<0.001), and was negatively correlated with ESR (r=-0.2862, P=0.0122), CRP (r=-0.2790, P=0.0184) and neutrophil cell counts (r=-0.287, P=0.011). SF DNase I activity was almost negative in patients with RA, ankylosing spondylitis (AS) and gouty arthritis (GA). SF cfDNA level in RA patients was significantly higher than that in patients with osteoarthritis (100.81±142.98 vs 18.98±31.40 μg/mL, P=0.002), but similar to that in patients with AS (45.85±47.67 μg/mL, P=0.428) and GA (162.95±97.49 μg/mL, P=0.132). In patients with inflammatory arthritis, SF cfDNA level was positively correlated with ESR (r=0.4106, P=0.0116) and CRP (r=0.5747, P=0.0002). Conclusion Impairment of DNase I activity may be responsible for the enhanced NETs generation and plays a role in the pathogenesis of RA.