Objective: Exploring the effect of oxysterol-binding protein-like protein 2 (OSBPL2) on the morphology and function of the actin cytoskeleton in auditory cells and its related molecular mechanisms. Methods: Osbpl2 knockout (Osbpl2-KO) HEI-OC1 cells was used to investigate the effect of OSBPL2 deficiency on the morphology and function of the actin cytoskeleton in auditory cells. Stereocilia morphology of hair cells in Osbpl2-KO mouse was observed by scanning electron microscopy. Western blot assay was used to detect the expression level of Rho-associated coiled-coil-forming kinase 2 (ROCK2), Lim Domain Kinase 1 (Limk1) and Actin depolymerization factor 1 (ADF/CFL-1) in HEI-OC1 cells and mouse cochleae. Results: The distribution of F-actin in the microfilament skeleton of Osbpl2-KO HEI-OC1 cells was significantly down-regulated, the peripheral microspikes and filopodia were significantly reduced, and the cell migration ability was significantly weakened. In vivo, Osbpl2 deficient resulted in shortened and disordered arrangement of stereocilia in mouse inner hair cells. Western blot assay showed that Osbpl2-KO in HEI-OC1 cells and mouse cochlea led to the inhibition of RhoA/ROCK2 signaling pathway. Conclusion: OSBPL2 is involved in the regulation of actin cytoskeleton as an important effector of RhoA/ROCK2 signaling pathway in auditory cells.