To identify the major serum biomarkers predicting the response to methotrexate (MTX) treatment in patients with early rheumatoid arthritis (RA), we evaluated the relationships between the individual response to MTX and various associated factors utilizing the (1)H nuclear magnetic resonance ((1)H NMR)-based metabolomic method. Thirty-eight early RA patients were enrolled in this cohort study, and they received MTX (10 mg/week) orally as monotherapy for 24 weeks. According to the American College of Rheumatology criteria for improvement, clinical evaluation following MTX treatment was carried out at baseline and at the end of 24 weeks. Furthermore, collected serum samples were analyzed using 600 M (1)H NMR for spectral binning. The obtained data were processed by both the unsupervised principal component analysis (PCA) and the supervised partial least squares discriminant analysis (PLS-DA). Lastly, multivariate analyses were performed to recognize the spectral pattern of endogenous metabolites related to MTX treatment. Differential clustering of (1)H NMR spectra identified by PCA was found between the effective (n=25) and non-effective (n=13) group of RA patients receiving MTX treatment. Multivariate statistical analysis showed a difference in metabolic profiles between the two groups using PLS-DA (R(2)=0.802, Q(2)=0.643). In targeted profiling, 11 endogenous metabolites of the effective group showed a significant difference when compared with those of the non-effective group (p<0.05). Serum metabolites correlated with MTX treatment in patients with early RA were identified, which may be the major predictive factors for evaluating the response to MTX treatment in patients with early RA. Furthermore, our results highlight the usefulness of (1)H NMR-based metabolomics as a feasible and efficient prognostic tool for predicting therapeutic efficacy to MTX treatment.