To elucidate the mechanism of invasion of oral squamous cell carcinoma, we newly established two different cell lines with a high-motility phenotype (designated HI type) and low-motility phenotype (LI type) from CA-9-22, a human oral squamous cell carcinoma cell line, through cell invasion assay (Boyden chamber assay). When we examined the subcellular localization and protein expression of actinin-4 using these cell lines, although the growth curves were not significantly different between the HI type and LI type, more invasion was seen in the HI-type than in the LI-type on Boyden chamber assay (p<0.0001). Morphologically, a larger number of sharply extended cell processes and spindle formation were observed in the HI-type than in the LI-type, and actinin-4 was mainly distributed in these processes. Western analysis showed that the expression level of actinin-4 was almost equivalent between the HI and LI types. These findings suggest that subcellular localization of actinin-4 might be involved in cell motility and cancer invasion by regulating the actin cytoskeleton at the cell processes in oral squamous cell carcinoma.