Cotton fiber development at the stages of elongation and secondary wall synthesis determines the traits of fiber length and strength. To date, the mechanisms controlling the progression of these two phases remain elusive. In this work, the function of a fiber-preferential actin-binding protein (GhPFN2) was characterized by cytological and molecular studies on the fibers of transgenic green-colored cotton (Gossypium hirsutum) through three successive generations. Overexpression of GhPFN2 caused pre-terminated cell elongation, resulting in a marked decrease in the length of mature fibers. Cytoskeleton staining and quantitative assay revealed that thicker and more abundant F-actin bundles formed during the elongation stage in GhPFN2-overexpressing fibers. Accompanying this alteration, the developmental reorientation of transverse microtubules to the oblique direction was advanced by 2 d at the period of transition from elongation to secondary wall deposition. Birefringence and reverse transcription-PCR analyses showed that earlier onset of secondary wall synthesis occurred in parallel. These data demonstrate that formation of the higher actin structure plays a determinant role in the progression of developmental phases in cotton fibers, and that GhPFN2 acts as a critical modulator in this process. Such a function of the actin cytoskeleton in cell phase conversion may be common to other secondary wall-containing plant cells.