ApoE plays a key role in various biological events. The aim of this study is to clarify its clinical significance in gastric cancer. We obtained paired clinical bulk samples of tumor tissue and corresponding normal tissue from 124 gastric cancer patients. To address ApoE mRNA expression clearly, we selected four samples, and differentially dissected gastric cancer and normal epithelium using laser microdissection (LMD) system. ApoE mRNA expression was examined by real-time reverse transcription (RT)-polymerase chain reaction (PCR). ApoE protein expression was assessed by immnunohistochemistry. The relationship between ApoE mRNA expression and clinicopathologic factors was statistically analyzed. RT-PCR assay for 124 bulk samples showed that ApoE mRNA expression was more highly expressed in gastric cancer tissue than in corresponding normal mucosa (p < 0.0001). By RT-PCR assay of four LMD samples, ApoE mRNA was overexpressed in gastric cancer. Immunohistochemistry showed that ApoE was predominantly expressed in gastric cancer. Tumors with high ApoE mRNA expression showed deeper tumor invasion into the muscle layer (p < 0.0001), the serosal layer (p < 0.01), or more positive lymph node metastasis (p < 0.05). When assessed by Kaplan-Meier analysis, patients with high ApoE expression tumor had a shorter survival than those with low ApoE expression tumor (p < 0.05). Moreover, multivariate analysis indicated that high ApoE mRNA expression was an independent indicator for muscular invasion (p < 0.01). ApoE is highly expressed in gastric cancer, contributing to shorter survival. In particular, ApoE was closely correlated with muscular invasion, and may be a possible biomarker predicting muscular invasion of gastric cancer.