25-Hydroxyvitamin D (25(OH)D) deficiency is a frequent condition in patients who suffer acute ischemic stroke (AIS), and several studies suggested that it may be associated with a poorer prognosis. Whether this association is affected by hypertension is unclear. Our aim was to investigate the association between 25(OH)D levels and both clinical severity and outcome after 3 months in AIS patients stratified by the history of hypertension. Consecutive first-ever AIS patients admitted to the Third Affiliated Hospital of Sun Yat-sen University, China were identified. Clinical information was collected. Serum 25(OH)D levels were measured at baseline. Stroke severity was assessed at admission using the National Institutes of Health Stroke Scale (NIHSS) score. Functional outcome was evaluated after 3 months of onset using the modified Rankin Scale (mRS). Multivariate analyses were performed using logistic regression models. During the study period, 377 patients were diagnosed as AIS and were included in the analysis. 25(OH)D deficiency was not associated with the risk of NIHSS at admission and 3 months mRS both in total patients and the hypertension subgroup. Among AIS without hypertension, 25(OH)D deficiency subjects had a significantly higher of NIHSS at admission and 3 months mRS compared with those with 25(OH)D ≥ 50 nmol/L. The odds ratios (95% confidence interval) were 5.51(1.83–16.60) and 4.63(1.53–14.05) in the multivariable adjusted model (P for linear trend < 0.05). Serum lower 25(OH)D levels can be seen as an independent prognostic factor of functional outcome in AIS without hypertension. Additional studies about improving prognosis of AIS by vitamin D supplementation could be first applied to these patients.