Several case reports have described associations between pathological nonvertebral fractures and low-dose methotrexate (MTX) in rheumatoid arthritis (RA) patients. Furthermore, a significant association between the C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene and incident fractures has been reported in postmenopausal women. We attempted to determine whether MTX use and MTHFR polymorphisms are associated with incident fracture risk in Japanese female RA patients. DNA samples, laboratory data, and clinical data were obtained from 731 female RA patients more than 50 years old as part of the Institute of Rheumatology Rheumatoid Arthritis (IORRA) observational cohort study. Genotyping of the MTHFR polymorphisms C677T and A1298C was performed using TaqMan SNP Genotyping Assays. MTX use, MTHFR polymorphisms, and other potential risk factors predictive of fracture were analyzed by Cox proportional hazards regression models, including time-dependent covariates. During 78 months from October 2000 to March 2007, 25 and 90 patients developed vertebral and nonvertebral fractures, respectively. Patients with nonvertebral fractures were more likely to take MTX (P = 0.011; odds ratio, 1.77; 95% confidence interval, 1.13-2.76) compared to patients without fractures. Although the C677T and A1298C polymorphisms were not significantly associated with incident fracture risk, MTX use, age, disease duration, and Japanese health assessment questionnaire score were significantly (P < 0.05) and independently associated with nonvertebral fracture incidence. Our results suggest that MTX use is associated with a nonvertebral fracture risk, whereas MTHFR polymorphism status does not appear to be a clinically useful marker for predicting fracture risk in Japanese female RA patients.