Methotrexate (MTX) is used widely as a first-line drug for the treatment of rheumatoid arthritis (RA) worldwide. There are large interindividual differences in the therapeutic response to MTX, but it is not known which factors influence them. We therefore investigated predictive factors associated with the therapeutic response to MTX in a hospital-based cohort study. Japanese adult RA outpatients prescribed MTX were enrolled and their characteristics were collected from the electronic medical records. The European League Against Rheumatism (EULAR) response criteria were used as the response to MTX therapy. The observation period was 1 year after beginning MTX administration. Sixteen types of single-nucleotide polymorphisms were investigated using the real-time PCR method. Associations between the MTX response and patient characteristics were evaluated using the multivariate logistic regression model. Among 70 Japanese adult RA outpatients, 52 were classified as MTX responders. In multivariate analysis, patients with the solute carrier family 19 member 1 ( SLC19A1 ) 80G > A A / A genotype had a better response than those with the A/G or G / G genotype, and patients with the C allele of γ- glutamyl hydrolase ( GGH ) 16T > C had a better response than those with the T / T genotype.This study showed that the therapeutic response to MTX in Japanese RA patients was associated with the genetic polymorphisms of SLC19A1 80G > A and GGH 16T > C in actual clinical practice.