Hormone D and its analogues display immunomodulatory activities providing a beneficial effect in immunoinflammatory diseases. The aim of this study was to assess the effect of alfacalcidol treatment on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity and glutathione (GSH) and malondialdehyde (MDA) levels in patients with active RA. Sixteen patients with active RA and twenty controls were enrolled in the study. Blood samples were taken before and after 12 weeks of alfacalcidol therapy (2 ?g/day). Oxidative stress parameters were determined spectrophotometrically and by flow cytometry assessment. Disease activity was assessed using DAS28 score. The results revealed that alfacalcidol treatment, significantly (p = 0.04) reduced SOD activity and CAT activity (p = 0.001) in RA patients. The activity of GPx was significantly lower in RA patients before treatment, compared to controls (p = 0.04). After therapy, GPx activity was restored to control levels, and GSH levels were significantly reduced (p = 0.01). MDA levels in patients at the beginning of the study protocol, remained significantly elevated compared to controls (p = 0.01). Alfacalcidol treatment decreased MDA levels in patients (p = 0.19). Furthermore, 12-weeks alfacalcidol therapy, changed the response of RA patients? PBMC to stimulation preventing the O2 - production and mitochondrial membrane depolarisation. After alfacalcidol treatment, significant clinical improvement was observed.