Objective This study aimed to investigate the predictive value of preoperative serum CA19-9 level for lymph node micrometastasis in patients with lymph node metastasis-negative gastric cancer and its effect on prognosis. Methods Clinicopathological data were retrospectively collected from 176 cases of gastric cancer who underwent D2 radical surgery in our hospital between January 2006 and December 2011, and also collected the patients’ lymph node tissue specimens. All patients were confirmed by pathologic examination of lymph node metastasis-negative. Quantitative real-time PCR (qRT-PCR) was used to detect the presence of lymph node micrometastasis in lymph node tissues. Sixty cases of gastric cancer were selected to construct the receiver operating characteristic curve (ROC) of preoperative serum CA19-9 level to predict lymph node micrometastasis, then established the threshold value. The remaining 116 cases were used to validate the rationality of this threshold. In addition, we explored the impact of preoperative serum CA19-9 level on the prognosis of patients with lymph node metastasis-negative gastric cancer, and explored the risk factors of lymph node micrometastasis. Results ① Results of ROC curve: the preoperative serum CA19-9 level of 15.5 U/mL was the threshold for predicting lymph node micrometastasis, with a sensitivity of 93.1%, specificity of 63.6%, and area under the curve (AUC) of 0.84 (P=0.003). With 15.5 U/mL as the threshold, 116 patients were divided into positive group and negative group. The lymph node micrometastasis rates in the 2 groups were different, which was higher in the positive group than that in the negative group (P<0.001). ② Effect of preoperative serum CA19-9 level on prognosis: the patients were divided into the positive group and the negative group with 15.5 U/mL as the threshold, and the log-rank test showed that the survival of the negative group was better than that of the positive group (P=0.001). ③ The risk factors for lymph node micrometastasis: the logistic regression model showed that preoperatively positive serum CA19-9 was an independent risk factor for lymph node micrometastasis in patients with gastric cancer [OR=1.860, 95% CI was (1.720, 2.343), P<0.001]. Conclusion Preoperative serum CA19-9 level can be used to predict lymph node micrometastasis in lymph node metastasis-negative patients with gastric cancer.