Abstract Objectives Extracellular vesicles (EVs) are abundant in body fluids, contributing to intercellular signalling by transferring cargo that includes microRNAs (miRs)—themselves implicated in pathobiology. For the first time we evaluated the potential of EV miRs to contribute diagnostic information in early RA, predict methotrexate (MTX) efficacy or shed light on the drug’s mechanism of action. Methods Seven hundred and ninety-eight miRs isolated from serum-derived EVs of 46 patients with untreated RA, 23 with untreated polymyalgia rheumatica (PMR; inflammatory disease control group) and 12 in whom significant inflammatory disease had been excluded (non-inflammatory controls; NICs) were profiled (NanoString); the same measurements were made for RA patients after 6 months’ MTX treatment. Analyses took multiple testing into account. Results Twenty-eight EV miRs were robustly differentially expressed between early RA (but not PMR) patients and NICs after correction for age and sex, suggesting discriminatory value. Cross-validated partial least squares-discriminant analysis also indicated the predictive potential of a distinct baseline EV miR signature with respect to MTX-induced remission at 6 months. The change in expression of 13 miRs over the course of MTX treatment differed significantly between responders and non-responders, and four of those exhibiting increased relative abundance amongst responders have known roles in regulating the pathogenic potential of synovial fibroblasts, namely miR-212-3p, miR-338-5p, miR-410-3p and miR-537. Conclusion Our data highlight the potential of serum EV miRs as diagnostic and therapeutic biomarkers, highlighting a novel potential mechanism by which MTX may exert its therapeutic effect in early RA that warrants further investigation.