Alemtuzumab is a highly effective treatment for multiple sclerosis (MS) and the treatment strategy of two cycles 12 months apart has a lot of appeal to patients. Widespread use of alemtuzumab has been tempered by treatment emergent autoimmunity which is seen in approximately one-third of patients in the 5 years after treatment. It has been postulated that relative vitamin D deficiency may be a causative factor in this setting. We have conducted a retrospective case-control study looking at the association of vitamin D and other potentially relevant clinical factors on the likelihood of treatment emergent autoimmune disease following alemtuzumab. Occurrence of autoimmunity was monitored for clinically and through the Bloodwatch® monitoring program. Clinical data and vitamin D levels obtained as part of routine clinical practice were recorded. Vitamin D levels were seasonally adjusted. Only cases with complete data were included. Univariable and multivariable Cox proportional hazards analyses were performed. There were 113 patients treated with alemtuzumab for whom there was complete data. Median follow up was 4.4 years. Risk of autoimmune disease was not associated with lower vitamin D levels. Risk of autoimmune disease was associated with female sex (HR 3.5) and with higher EDSS score at treatment. The association with EDSS was lost when analysis was restricted to those with 4 or more years of follow up. These data do not support a role for vitamin D supplementation in the prevention of autoimmune disease following alemtuzumab. Males have a lower risk of autoimmunity following alemtuzumab.