The polarized distribution of F-actin is important in providing the driving force for directional migration in mammalian leukocytes and Dictyostelium cells, in which compartmentation of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol phosphatase is critical for the establishment of cell polarity. Since monospores from the red alga Porphyra yezoensis are a real example of migrating plant cells, the involvement of the cytoskeleton and PI3K was investigated during their early development. Our results indicate that the asymmetrical localization of F-actin at the leading edge is fixed by the establishment of the anterior-posterior axis in migrating monospores, which is PI3K-dependent and protein synthesis-independent. After migration, monospores adhere to the substratum and then become upright, developing into multicellular thalli via the establishment of the apical-basal axis. In this process, F-actin usually accumulates at the bottom of the basal cell and development after migration requires new protein synthesis. These findings suggest that the establishment of anterior-posterior and apical-basal axes are differentially regulated during the early development of monospores. Our results also indicate that PI3K-dependent F-actin asymmetry is evolutionally conserved in relation to the establishment of cell polarity in migrating eukaryotic cells.