•The largest ambispective study from a developing country on natalizumab and multiple sclerosis.•Natalizumab was found to be highly effective in Indian patients with MS.•Patients with RRMS and SPMS improved in relapse rate while EDSS improvement was observed only in the RRMS cohort.•The risk-benefit ratio appears highly favorable with no reports of PML. Natalizumab (NTZ) is increasingly being used in Indian multiple sclerosis (MS) patients. There are no reports on its safety and efficacy, especially with respect to the occurrence of progressive multifocal leukoencephalopathy (PML). To describe the patient characteristics, treatment outcomes, and adverse events, especially the occurrence of PML in NTZ-treated patients. A multicentre ambispective study was conducted across 18 centres, from Jan 2012 to Dec 2021. Patients at and above the age of 18 years treated with NTZ were included. Descriptive and comparative statistics were applied to analyze data. During the study period of 9 years, 116 patients were treated with NTZ. Mean age of the cohort was 35.6 ± 9.7 years; 83/116 (71.6%) were females. Relapse rate for the entire cohort in the year before NTZ was 3.1 ± 1.51 while one year after was 0.20±0.57 (p = 0.001; CI 2.45 -3.35). EDSS of the entire cohort in the year before NTZ was 4.5 ± 1.94 and one year after was 3.8 ± 2.7 (p = 0.013; CI 0.16–1.36). At last follow up (38.3 ± 22.78 months) there were no cases of PML identified. Natalizumab is highly effective and safe in Indian MS patients, with no cases of PML identified at last follow up.