The main objective of this study was to investigate the prevalence of insufficient serum 25-hydroxyvitamin D level in a sample of neurological patients and to compare it to the estimate in the general Croatian population. The secondary aim was to test for the possible significant differences in vitamin D2 insufficiency between the groups of patients according to gender, season, region, vitamin D3 supplementation, and diagnosis of autoimmune disease. This retrospective study involved 371 neurological patients in one-year period. The data collected included gender, age at the time of admission, region, season at the time of admission, main neurological diagnosis at discharge, serum 25-hydroxyvitamin D level at the time of hospitalization, and use of vitamin D3 substitutes prior to hospitalization. The proportion of neurological patients with 25-hydroxyvitamin D insufficiency (<75 nmol/L) was estimated at 74.66% (95% confidence interval, p<0.0001), which is slightly lower than in the Croatian general population. There were no significant differences in 25-hydroxyvitamin D insufficiency according to gender, season and region. Significant differences in 25-hydroxyvitamin D insufficiency were found according to vitamin D3 supplementation and diagnosis of autoimmune disease.