Objective To detect the serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in the elderly patients with gastric cancer, and further explore the correlations of the level with the clinical features and prognosis of the disease. Methods A total of 65 elderly patients (ranging 62 to 83 years old) with newly diagnosed gastric cancer pathologically in our hospital from September 2009 to September 2012 were enrolled in this study. Their morning fasting blood samples before and after treatment were collected for the determination of serum level of 1,25-(OH)2D3 by enzyme-linked immunosorbent assay (ELISA). The correlations of the levels with clinical pathological features and prognosis were analyzed. Results The original level of 1,25-(OH)2D3 was (18.26±4.13)μg/L in these 65 patients after pathological diagnosis before any treatment, and the advanced level became (9.26±3.21)μg/L after treatment, with statistical difference (P＝0.028). The original and advanced levels were significantly correlated with clinical stages and cell differentiation (P＜0.05), but not with tumor locations, depth of infiltration, lymph node metastases and distant metastases (P＞0.05). The patients with high levels of 1,25-(OH)2D3 (＞20μg/L) had a higher overall survival rate compared with those with lower levels (≤20μg/L; P＜0.05). Conclusion Serum level of 1,25-(OH)2D3 might be used as an prognostic index for gastric cancer of the elderly patients.