Objective  To investigate the effects of Rho-associated coiled-coil-forming kinase（ROCK）inhibitor Y27632 on the development of mouse preantral follicles in vitro.    Methods  The ovaries of 12.5-day-old mice were collected and single preantral follicle was isolated by mechanical method  to culture Nunclon Sphera 96-well U-bottom 3D cell culture plate in vitro. Set up a control group and treated group containing Y27632, and evaluate the overall development of the follicles through follicular morphology, follicle diameter, the number of mature follicles, and changes in oocyte spindles. The expression of oocytes ［bone morphogenetic protein 15(BMP15), growth differentiation factor 9(GDF9)］, granulosa cells ［follicle stimulating hormone receptor (FSHR)］ and apoptosis related genes (Bad, Bax and Caspase-3) were also assessed by the Real-time PCR technique. Meanwhile, the follicle viability was detected by follicular activity test during follicular development.    Results  Rock inhibitor Y27632 had no significant effect on the growth diameter and basic development indicators (follicle survival rate, cavity formation rate and maturation rate) (P>0.05). And abnormal spindle assembly occurred during follicle development in the control group. After 8 days of culture, compared with the control group, the granulosa cell-specific gene FSHR was up-regulated in the experimental group; As well as the oocyte-specific genes BMP15 and GDF9 were up-regulated, while forkhead box O3(FoxO3) was down-regulated; The apoptosis genes Bax, Bad and Caspase-3 were also showed down-regulated. And the granulosa cell apoptosis in the experimental group was significantly less than that in the control group.    Conclusion  ROCK inhibitor Y27632 can inhibit the apoptosis of granulosa cells and prevents the abnormal assembly of oocyte spindles to improve the quality of follicle development in vitro.