Abstract: Objective To investigate the association between serum 25-(OH) D3, Ca²⁺ and gastric cancer. Methods A total of 63 patients pathologically diagnosed as chronic superficial gastritis(Control group) and a total of 118 patients pathologically diagnosed as gastric cancer(Case group) were selected from The First Affiliated Hospital of Zhengzhou University. The level of serum 25-(OH) D3 was measured by ELISA, and the level of Ca²⁺ was measured by ISE potential method. Results The level of 25-(OH) D3 of case group was significantly lower than that of control group(t=15.07, P=0.000), but no significance was found in comparison of Ca²⁺ between them(t=1.109, P=0.269). There was a positive correlation between serum 25-(OH) D3 concentration and Ca²⁺ in case group(r=0.706, P=0.000). The difference of 25-(OH) D3 levels among case groups with different cell differentiation degrees was statistically significant(F=3.356, P=0.038). When patients had taken gastric operation, the serum level of 25-(OH) D3 was higher in postoperative group than that in preoperative group（t=−8.017, P=0.000）. However, no statistical significance was found between positive lymph node metastasis group and negative lymph node metastasis group (t=0.061, P=0.952). Conclusion The level of serum 25-(OH) D3 may be associated with the occurrence and development of gastric cancer, and it is significantly increased after surgery; therefore, the serum 25-(OH) D3 could be used to monitor gastric carcinoma patients' conditions, prediction and prognosis.