This study aims to investigate the correlation between serum 25-hydroxyvitamin D3 [25 (OH)D3] concentration and depressive state in stroke patients aged 65 years and above. Methods: Relevant data of elderly stroke patients (≥65 years old) were collected from the National Health and Nutrition Examination Survey (NHANES), and depressive state was assessed according to the results of the Patient Health Questionnaire 9-item (PHQ-9). Patients were divided into non-depressed and depressed groups based on these assessments. Differences in clinical data between the two groups were compared, and a multivariate Logistic regression model was used to evaluate the correlation between serum 25(OH)D3 concentration and depressive state. Results: A total of 600 stroke patients aged ≥65 years were included, of which 76 cases (12.67%) were included in the depression group. The age and serum 25(OH)D3 concentration of patients in the depression group were lower than those in the non-depression group (both P<0.05), and the proportion of marital status being married or cohabiting was higher than that in the non-depression group (P<0.05). Multivariate Logistic regression analysis showed that increased serum 25(OH)D3 concentration was negatively correlated with the risk of depressive symptoms (OR=0.917, 95%CI 0.844-0.997, P=0.042). Conclusion: The increase in serum 25(OH)D3 concentration is negatively correlated with the risk of depressive state in elderly stroke patients, suggesting that routine screening of serum 25(OH)D3 should be recommended for this population.