Objective To evaluate the efficacy and toxicity of methotrexate (MTX) in the treatment of ankylosing spondylitis (AS). Methods Randomized controlled trials (RCTs) were identified from CENTRAL (The Cochrane Library Issue 4, 2005); MEDLINE (1966 to November 2005); EMBASE (1980 to November 2005); CINAHL (1982 to November 2005). The quality of included trials was evaluated. Data were extracted by two reviewers independently using a specially designed extraction form. The Cochrane Collaboration’s RevMan 4.2 software was used for data analysis. Results Three trials involving 116 patients were included. One 12-month trial compared naproxen plus MTX with naproxen alone. Two 24-week trials compared different doses of MTX with placebo. No statistically significant differences were found for the primary outcome measures of physical function, pain, spinal mobility, peripheral joints/entheses pain, swelling and tenderness, changes in spine radiographs and patient and physician global assessment. The response rate in one trial showed statistically significant benefits of 36% in the MTX group compared with the placebo group (RR 3.18, 95% CI 1.03 to 9.79). This response rate was a composite index including assessment of morning stiffness, physical well-being, Bath ankylosing spondylitis disease activity index (BASDAI), Bath ankylosing spondylitis functional index (BASFI), health assessment questionnaire for spondyloarthropathies (HAQ-S), and physician and patient global assessment. However, no single outcome showed a statistically significant difference between the MTX and placebo groups when endpoint results were compared. Therefore, this benefit of MTX was questionable. No serious side effects were reported in these studies. Conclusions There is no enough evidence to show any benefit of MTX in the treatment of AS. High quality randomized controlled trials of longer duration and with larger sample size are needed to clarify the effect of MTX on AS.