Objective To explore regularity of lymph node metastasis and analyze its relation between lymph node metastasis and histological features and its immunohistochemical markers of gastric cancer, and to provide evidence for selection of reasonable operation. Method The clinical data of 160 patients with gastric cancer who underwent D2, D3 or D3+ from August 2013 to May 2016 in the Second Hospital of Lanzhou University were retrospectively studied, and the relation between the lymph node metastasis and the pathological features and the immunohistochemical markers in the different location of gastric cancer was analyzed. Results ① The rate of lymph node metastasis in the early gastric cancer was significantly lower than that in the advanced gastric cancer (P<0.05), which in the T4 stage was significantly higher than that in the T1–T3 stages (P<0.05), in the poorly differentiated gastric cancer was significantly higher than that in the well differentiated gastric cancer (P<0.05), or in the Borrmann type Ⅲ+Ⅳ (infiltrative type) was significantly higher than that in the Borrmann type Ⅰ+Ⅱ (topical type,P<0.05), but which wasn’t associated with the gender, tumor location, or tumor diameter (P>0.05). ② The lymph node metastasis occurred mainly in the first and the second stations for the well differentiated gastric cardia cancer, which not only occurred in the first and the second stations, but also occurred in the No.13 lymph node for the poorly differentiated gastric cardia cancer; which occurred mainly in the first and the second stations and occasionally occurred in the No.12 lymph node for the well differentiated gastric body cancer, which not only occurred in the first and the second stations, but also occurred in the No.12, No.13 and No.14 lymph nodes for the poorly differentiated gastric body cancer; which occurred in the No.11, No.12 and No.13 lymph nodes for the part of well differentiated gastric antrum cancer, which even occurred in the No.15 and No.16 lymph nodes for the part of poorly differentiated gastric antrum cancer. ③ The expression positive rates of the TopoⅡα, Villin, Ki-67, CK-8, and CK-18 proteins in the poorly differentiated gastric cancer were significantly higher than those in the well differentiated gastric cancer (P<0.05), which of the P-gp, GST-π, and c-erbB-2 proteins in the poorly differentiated gastric cancer were significantly lower than those in the well differentiated gastric cancer (P<0.05). The expression positive rates of the TopoⅡα, P-gp, Villin, Ki-67, CK-8, and CK-18 proteins in the gastric cancer with lymph node metastasis were significantly higher than those in the gastric cancer without lymph node metastasis (P<0.05), whereas there were no relation between the expression positive rates of the GST-π and c-erbB-2 proteins and the lymph node metastasis of gastric cancer (P>0.05). ④ The different location of gastric cancer wasn’t associated with the gender, gross type, clinical stage, T stage, degree of differentiation, Borrmann type, or tumor diameter. Conclusions In advanced gastric cancer, depth of tumor invasion reached T4, poor degree of differentiation, and Borrmann infiltration type of gastric cancer, lymph node metastasis rates are higher. For gastric cardia cancer patients with well differentiation, standard D2 should be performed, D2+No.13 should be performed for poor differentiation. For gastric body cancer patients with well differentiation, D2+No.12 should be performed, D3 should be performed for poor differentiation. For gastric antrum cancer patients with differentiation degree or not, D3 should be performed, selective dissection of No.15 or No.16 lymph node should be performed for poor differentiation. Combined detection of TopoⅡα, Villin, Ki-67, CK-8, CK-18, P-gp, GST-π, and c-erbB-2 immunohistochemical markers might be helpful to improve accuracy of lymph node metastasis and evaluate degree of malignancy and prognosis of patients with gastric cancer.