Objective：To explore the effect of oxysterol-binding protein-like 2(OSBPL2)on the actin cytoskeleton in auditory cells and its related molecular mechanisms. Methods：OSBPl2 knockout(Osbpl2-KO)HEI-OC1 cells were used to investigate the effect of OSBPL2 deficiency on the the actin cytoskeleton in auditory cells. The stereocilia onmouse hair cells were observed by scanning electron microscopy. Western blot was used to detect the expression of Rho - associated coiled - coil - containing protein kinase 2 (ROCK2)，Lim domain kinase 1(LIMK1)and actin depolymerizing factor 1(ADF/CFL - 1)in HEI -OC1 cells and mouse cochleae. Results：In Osbpl2-KO HEI-OC1 cells，the distribution of F-actin in the microfilament skeleton was down-regulated，the pericellular microspikes and filopodia were noticeably reduced，and the cell migration ability was significantly weakened. In vivo，OSBPL2 deficiency resulted in shortened and disordered stereocilia on mouse inner hair cells. Western blot assay showed that OSBPL2 deficiency led to the inhibition of RhoA/ROCK2 signaling pathway in HEI - OC1 cells and mouse cochleae. Conclusion：OSBPL2 mediats RhoA/ROCK2 signaling pathway in auditory cells and plays an important role in maintaining the morphology and function of actin cytoskeleton.