Abstract Objective We aim to evaluate 3-year effects of ocrelizumab (humanized anti-CD20 monoclonal antibody for the treatment of multiple sclerosis (MS)) on lymphocytes, neutrophils and immunoglobulins: (1) when compared with pre-infusion assessment; (2) over the course of treatment; and (3) possible clinical correlates of the observed immunological modifications. Methods This real-world observational cohort study has been conducted on prospectively collected data from 78 MS patients (mean age 47.8 ± 10.5 years; females 48.7%) commencing on ocrelizumab from 2018, with mean follow-up of 36.5 ± 6.8 months. Clinical data and blood samples were collected every three months. Total lymphocyte count and subpopulations were assessed on peripheral blood using flow cytometry. Serum immunoglobulins were evaluated with nephelometry. Results When compared with pre-infusion values, we observed reduction of total, CD19 and CD20 lymphocyte counts; however, after the first infusion, their levels remained substantially stable. Over time we observed a progressive reduction of CD8 lymphocytes, while no changes were observed for CD4, CD27, CD3CD27, and CD19CD27. After the first infusion, we observed reduction in IgG, which further decreased during the follow-up. Higher probability of EDSS progression was associated with reduced modulation of CD8 lymphocytes. Interpretation Ocrelizumab affects both humoral and cellular immune responses. Disability progression over the follow-up was associated with lower CD8 cytotoxic T-lymphocyte reduction. Changes in humoral response are immediate and sustained, while modulation of cellular immunity occurs progressively through regular re-treatment, and is related to clinical stability.