The objective of this study is to investigate lysosome-associated protein transmembrane 4b-35 (LAPTM4B-35) protein expression in gastric cancer and its clinical implications. LAPTM4B-35 protein expression in 652 gastric cancer specimens and matched adjacent non-cancerous tissues was evaluated by immunohistochemical staining. Subsequently, the relationship between LAPTM4B-35 protein expression, clinical pathological parameters, and prognosis of gastric cancer was determined. LAPTM4B-35 protein expression was significantly higher in gastric cancer tissue compared to adjacent non-cancerous tissues (P = 0.001). In total, 417 (63.96 %) of the 652 gastric cancer cases demonstrated high LAPTM4B-35 protein expression. Multiple regression analysis was performed to identify the factors related to lymph node metastasis. As a result, age, tumor number, primary tumor category, histological type, histological growth pattern, and LAPTM4B-35 protein expression were found to be significantly related to lymph node metastasis (P = 0.010, 0.001, 0.032, 0.001, 0.001, and 0.001, respectively). Survival analysis identified that LAPTM4B-35 protein expression was also an independent prognostic factor (P = 0.001). LAPTM4B-35 was highly expressed in gastric cancer and may be a potential target for the management of gastric cancer.