Natalizumab is one option for multiple sclerosis patients responding poorly to classical immunomodulators, but pilot studies did not point to its effectiveness as a second-line therapy. Aim of this study was to assess the efficacy of natalizumab as second-line therapy in patients switching from disease modifying therapies (DMTs) in a clinical setting. We retrospectively selected patients who had been treated with natalizumab for at least 12 months after switching from one or more DMTs. We collected clinical and neuroradiological data and we analysed the reduction in annualised relapse rate (ARR), the change of Expanded Disability Status Scale (EDSS) and the reduction of contrast-enhancing lesions (CELs) at magnetic resonance imaging (MRI) of the brain at 12 months of natalizumab and of previous DMT therapy. Fifty patients were included in the analysis (11 males, 39 females).We observed a reduction of ARR on natalizumab (p = 0.000) and a statistically significant different trend of relapse event between the two treatments (p = 0.0149). EDSS was stable during natalizumab therapy whilst it showed an increase on DMTs (p = 0.0244). The number of CELs decreased significantly (p = 0.006) during the 12 months of treatment with natalizumab, whilst it was stable on DMTs. Natalizumab showed to decrease ARR, stabilize EDSS, increase the percentage of CELs free patients and decrease the number of CELs in a group of 50 poor responders to classical DMT, after the first 12 months of therapy.