Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, but the evidence to conclusively prove this hypothesis remains uncertain. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC-rich population. The aim of this study was to clarify the characterization of the SP cells in peritoneal metastasis of gastric carcinoma. Gastric cancer cell lines OCUM-2M, OCUM-2D, and OCUM-2MD3 (a daughter cell line with high potential for peritoneal metastasis) were used. We isolated SP cells from OCUM-2M and OCUM-2D using flow cytometry. Serial sorting was performed three times to enrich SP cells, and they were designated as OCUM-2M/SP and OCUM-2D/SP cells. Flow cytometric analysis showed 0.46%, 0.29%, 5.24%, 6.49%, and 11.3% of the SP cells to be found in OCUM-2M, OCUM-2D, OCUM-2MD3, OCUM-2M/SP, and OCUM-2D/SP cells, respectively. The intraperitoneal inoculation of SP cells and OCUM-2MD3 cells produced peritoneal metastasis, but parent cells did not. The adhesion ability of SP and OCUM-2MD3 cells was significantly high in comparison to that of parent cells. The expression level of adhesion molecules alpha2-, alpha5-, beta3-, and beta5-integrin, and CD44, was high in SP cells compared to parent cells. The expression of stemness markers, Oct3/4 and Sox2, increased in the SP-cell-injected tumors. These findings suggested that CSC-like SP cells expressing alpha2-, alpha5-, beta3-, and beta5-integrin, and CD44, may play an important role for peritoneal metastasis in gastric carcinoma. Oct3/4 and Sox2 may be associated with CSC in gastric cancer.