Neurons send out their axons that carry information to distant target cells. Outgrowth, branching and guidance of axons are very important steps to establish the functional neuronal circuits. We previously reported that R-Ras, a Ras-family small GTPase, has essential roles in axon specification and guidance. Furthermore, we have revealed that afadin, an actin-binding protein, functions as an effector molecule of R-Ras and regulates axon branching. Afadin has been known to comprise two isoforms, l-afadin and s-afadin; l-afadin, the long form of afadin, has two Ras-association (RA) domains, a forkhead-associated (FHA) domain, a DIL domain, a PDZ domain, three proline-rich (PR) domains and an F-actin-binding domain while s-afadin, the short form of afadin, lacks the F-actin-binding domain and the third proline-rich domain. l-afadin is expressed in various tissues including lung, kidney and brain but s-afadin is specifically expressed in brain. However, the function of s-afadin in brain remains poorly understood. In this study, we tried to elucidate the functions of l-afadin and s-afadin in axonal morphology, respectively. We examined expression pattern of each afadin isoform in neuronal developmental stages in cultured cortical neurons and we found that the expression of l-afadin was high during all axon developmental stages and s-afadin was gradually expressed in later stage. RNA interference knockdown of l-afadin suppressed axon branching, while that of s-afadin promoted axon branching. Immunoprecipitation assay showed that both of l-afadin and s-afadin bound to active R-Ras. These results suggest that both of l-afadin and s-afadin receive signal from R-Ras as downstream effectors and the former positively regulates R-Ras-mediated axon branching through binding to actin filaments while the latter negatively regulates the axon branching. Therefore, two types of afadin induce opposite control of axon morphology through differential regulation of R-Ras-mediated axon branching.