Actin dynamics are essential for root hair development, however, the underlying molecular mechanisms of actin binding protein cooperation and plant abiotic stress responses are largely unknown. Here, genetic analysis displayed that actin depolymerizing protein ADF7 and actin bundling protein VLN1 are positively and negatively involved in root hair development in Arabidopsis, respectively. Moreover, ADF7 acts upstream of VLN1 in root hair development by the analysis of RT-qPCR, Gus staining, Western blot and genetics. The observation of F-actin dynamics shows that ADF7 inhibits VLN1, leading to the decline of filament actin (F-actin) bundling and thick bundle formation and the increase of F-actin turnover and depolymerization in epidermal cells of root apices. Actin pharmacological experiments confirm that ADF7 and VLN1 are via regulating F-actin dynamics to active root hair development. Furthermore, F-actin depolymerization coregulated by ADF7 and VLN1 elevates the reactive oxygen species (ROS) level in root tips. Additionally, F-actin depolymerization and ROS accumulation coregulated by ADF7 and VLN1 are involved in osmotic stress-induced root hair development. Our work reveals that ADF7 inhibits VLN1 to induce F-actin turnover and depolymerization and ROS level in root tips, which play an important role in root hair formation responses to osmotic stress .