Objective To investigate the association of Mycoplasma pneumoniae(MP) infection with disease activity of ankylosing spondylitis. Methods A total of 158 subjects in our hospital were enrolled in this study, including patients with ankylosing spondylitis(AS, n=66), rheumatoid arthritis (RA, n=31),osteoarthritis(OA, n=25) and normal controls(NC, n=36). MP infection was defined as anti-MP IgM antibody positive. Anti-MP IgM antibodies were determined by a mycoplasma pneumoniae(Mac strain)membrane-based agglutination test. AS patients were divided into two groups: MP infection group and non-MP infection group. T-test was used for statistical analysis of age, blood white cells, ESR, CRP, immunoglobulin, BASDAI index, global assessment on VAS scale, Schober test and chest expansion reflecting spinal mobility.χ2-test was used to compare the positive rate of MP infection in different groups. Gender difference and prevalence of clinical infection in past four weeks between MP infection and MP-free group in AS patients was also compared. Ridit analysis was used to analyze the association of MP infection with degree of sacroiliac damage on CT. Results The prevalence of MP infection in AS (52%, 34/66) was much higher than that in rheumatoid arthritis (RA, 6%, P＜0.01 ), osteoarthritis(OA, 4%, P＜0.01 ) and normal controls (NC, 11%, P＜0.01) . Compared with the non-MP infection group, the MP infection group had more active disease in term of BASDAI(4.0±1.1 vs 3.0±1.9, P=0.017), ESR[(44±32) mm/1h vs (28±23) mm/1h, P=0.029], CRP [(40±38) mg/L vs (22±21) mg/L, P=0.025] serum total IgG level [(18±3) g/L vs (16±5) g/L, P=0.027],but not in serum total IgA and IgM. Regarding to the sacroiliac joint and spinal mobility, MP infection group did not exhibit any association with the sacroiliac grading on CT, Schober test and expansion. In AS patients with MP infection, only 44.1%(15/34) was complicated by clinical manifestations of upper respiratory tract in the past 4 weeks. However, a higher prevalence of MP infection was found in AS patients with clinical manifestation of upper respiratory tract, compared with those with negative clinical manifestation(71% vs 42%,P=0.027). Conclusion Mycoplasma pneumoniae is the most common reported pathogen in ankylosing spondylitis and relates to the disease activity of AS. MP infection is probably a principal triggering factor in the pathogenesis of AS.