This study aims to measure the serum level of 25-hydroxyvitamin D₃ (25-OH-D₃) in 302 patients with rheumatoid arthritis (RA), studying the association to disease activity. Three hundred two RA patients underwent clinical examination and serological analysis. 25-Hydroxyvitamin D₃ was determined by high-performance liquid chromatography-tandem mass spectrometry. Vitamin D₃ deficiency defined as serum levels of 25-hydroxyvitamin D₃ below 50 nmol/l was detected in 101 RA patients (33.4 %). There was no significant correlation between the serum level of 25-hydroxyvitamin D₃ and Disease Activity Score 28 (DAS28) (3w) score. In a subpopulation of RA patients with very low serum level of 25-OH-D₃ (≤15 nmol/l) (n = 15), there were significant differences compared to patients with normal 25-OH-D3 (n = 200): higher percentage of patients with positive rheumatoid factor (100.0 versus 77.5 %; p = 0.05), higher CRP (28.7 versus 14.8 mg/l; p = 0.001), higher number of patients treated with at least three disease-modifying antirheumatic drugs (DMARDs) (40.0 versus 14.5 %; p = 0.02), higher number of patients with high disease activity DAS28 score of ≥5.1 (20.0 versus 4.5 %; p = 0.01), lower age (54.5 versus 64.0 years; p = 0.003) and shorter disease duration (5.1 versus 10.3 years; p = 0.06). Deficiency of 25-hydroxyvitamin D₃ was detected in 33.4 % of the RA patients. A subpopulation of patients with severe deficiency of vitamin D₃ serum level of ≤15 nmol/l was characterised by all being positive for rheumatoid factor, high percentage of patients with very high disease activity and high percentage of patients treated with at least three DMARDs.