CD44 is a cell surface protein and it is widely used as a cancer stem cell marker in various cancer types including gastric cancer. We conducted proteomic analysis in CD44(+) and CD44(-) gastric cancer cells to understand characteristics of CD44(+) and CD44(-) cells. In the present study, we sorted cells from the gastric cancer cell line MKN45 according to CD44 expression to separate out CD44(+) and CD44(-) cells. And we conducted RT-PCR to identify mRNA expression of cancer stem cell markers in CD44(+) and CD44(-) cells. Cancer stem cell markers showed upregulated expression in CD44(+) cells. Next, we performed two-dimensional electrophoresis analysis to determine the differential expression pattern of proteins in each group; control, CD44(+), and CD44(-) MKN45 cells. We found a total of 113 spots that varied in expression between CD44(+) and CD44(-) cells, and subjected 20 of those protein spots to MALDI-MS. We selected the three proteins (HSPA8; heat shock cognate 71 kDa protein isoform 1, ezrin, α-enolase) upregulated in CD44(+) cells than CD44(-) cells and one protein (prohibitin) showed increased expression in CD44(-) cells. We validated the protein expression levels of four selected proteins by Western blot. We suggest that our study could be a helpful background to study CD44(+) cancer stem-like cells and differences between CD44(+) and CD44(-) cells in gastric cancer.