Vitamin D (VD) plays a regulatory role in tumor occurrence and development, although the factors influencing serum 25-hydroxyvitamin D3 (25(OH)D3) levels in patients with cancer have not been studied. Therefore, we aimed to evaluate circulating levels of 25(OH)D3 and factors influencing the VD status in patients with malignant tumors. Adult patients with malignant tumors who had undergone assessments of serum 25(OH)D3 at Beijing Cancer Hospital from January 2019 to July 2022 were included. A multiple logistic regression model was applied to explore the associations of patient characteristics, environment, and disease characteristics with 25(OH)D3 levels. Among the 1,076 included patients, the median 25(OH)D3 serum concentration was 16.25 ng/mL. VD deficiency and the combined VD insufficiency and sufficiency were observed in 811 (75.37%) and 265 (24.63%) patients, respectively. Latitude, season, sex, body mass index, and type of cancer were associated with VD concentration/status in patients with malignant tumors. 25(OH)D3 concentrations were significantly higher in patients with thyroid cancer and significantly lower in patients receiving tumor-related treatment than in untreated patients. Surprisingly, we observed 25(OH)D3 serum concentration was lower in patients receiving nutritional supplementation than in those receiving no nutritional supplements. Patients with malignant tumors are at high risk of VD deficiency.