Gastric cancer (GC) is one of the most common human cancers. Spheroid colony formation is an effective model for characterization of cancer stem cells. However, gene expression profiles of spheroid colonies obtained from GC cells have not been examined. We performed microarray analyses by Human Genome U133 Plus 2.0 Array in spheroid body-forming and parental cells from MKN-45 and MKN-74 GC cell lines. Kinesin family member C1 (KIFC1) was expressed >2-fold higher in spheroid body-forming cells than in parental cells in both GC lines. Both the number and size of spheres from MKN-45 cells were significantly reduced upon KIFC1 siRNA-transfection compared with negative control siRNA-transfection. Immunohistochemical analysis of 114 GC tissue samples revealed that 42 (37%) of GC cases were positive for KIFC1 expression. GC cases positive for KIFC1 were found more frequently in stage III/IV cases than in stage I/II cases. GC cases positive for KIFC1 were found more frequently in intestinal type GC cases than in diffuse type GC cases. Furthermore, KIFC1-positive GC cases showed high Ki-67 labeling index. Kaplan-Meier analysis demonstrated that KIFC1 expression was not associated with survival. We found positive expression of KIFC1 in CD44‑positive GC and aldehyde dehydrogenase 1 (ALDH1)-positive GC cells. Our results showed that KIFC1 is overexpressed in GC. Since knockdown of KIFC1 inhibited sphere formation, KIFC1 likely plays an important role in cancer stem cells.