Low levels of serum vitamin D are common in patients with mood disorders and stroke. It has been shown that low levels of serum vitamin D indicate a risk of depression in post-stroke subjects. Our aim was to determine the relationship between vitamin D and post-stroke anxiety (PSA).A consecutive series of 226 first acute ischemic stroke patients were recruited and followed up for 1 month. Serum levels of vitamin D were measured within 24 hours of admission. Patients with significant clinical symptoms of anxiety and a Hamilton anxiety scale score >7 were diagnosed as having PSA. In addition, 100 healthy subjects were recruited as controls and underwent measurements of serum vitamin D.A total of 60 patients (26.55%) showed anxiety at 1 month. Both PSA patients and non-PSA patients had lower serum levels of vitamin D than healthy subjects. A significant relationship was found between PSA and serum levels of vitamin D. Low serum levels of vitamin D (≤38.48 nmol/L) were independently associated with the development of PSA (OR: 2.49, 95% CI: 1.21-5.13, P = 0.01).Serum vitamin D status is related to the occurrence of anxiety in post-stroke patients and may be an independent risk factor of PSA after 1 month.