There is growing evidence for crosstalk between septin filaments and actin cytoskeleton which is regulated by Rho family of GTPases. Here we show that active Rho disrupts septin filament structures in rat embryonic fibroblast REF52 cells. Among Rho effector molecules tested, Rhotekin induced morphological changes of septin filaments similar to those by activated Rho. The center region of Rhotekin was sufficient for the septin reorganization in the cells, and likely to interact indirectly with the C-terminal half of a septin Sept9b, where a GTPase domain is located. Rhotekin and Sept9b are colocalized mainly in perinuclear regions in serum-starved REF52 cells. Upon stimulation with lysophosphatidic acid, they translocated to actin stress fibers in 10 min and then redistributed again to cytoplasm after 90 min treatment. In neuroblastoma Neuro2a cells, Rhotekin and Sept9b were enriched in the tip of neurites, a location where cortical actin reorganization is induced upon stimulation with lysophosphatidic acid. Taken together, we propose that Rhotekin is a novel regulator organizing mammalian septin structures and provide a new link between the septin and Rho-signaling.