Actin, a cytoskeletal protein, gathers and polymerizes under the cell membrane to mediate protrusion of the cell membrane. Therefore, actin participates greatly in cell movement. To quantitatively evaluate actin's dynamics during cell protrusion, movements of actin-labeled cells were observed under a confocal laser scanning microscopy, and time-lapse images were obtained. Image analysis of the orientation of actin stress fibers revealed dynamic formation of a meshwork of stress fibers in the protrusion structure at the leading edge of the motile cell. In addition, a number of immobile spots such as focal adhesions were observed adjacent to the dynamic meshwork of stress fibers. The distribution and the time between appearance and disappearance of each spot were analyzed. The results suggest that immobile spots close to the centroid of the cell play a crucial role in providing anchorage and driving cell movement. © 2014 The Institute of Electrical Engineers of Japan.