The mechanisms underlying chemotherapy-induced acceleration of ovarian insufficiency are not fully understood, particularly for ovarian granulosa cells (GCs). We used two widely used cancer chemotherapeutic reagents, bleomycin and VP-16, and an in vivo GC-specific DNA topoisomerase II-β (TOP2β) (Top2b) knockout mouse model to investigate the effects of chemotherapy-induced DNA damage on growing mouse follicles. Bleomycin and VP-16 caused massive double-strand DNA breaks in the GCs of growing follicles in a time-dependent manner as shown by DNA-damage checkpoint activation. This damage was associated with apoptotic GC death and resulted in follicle atresia and ovulation failure. However, FSH-regulated ovarian functions, including estrogen biosynthesis and estrogen target gene expression, were not significantly affected by these genotoxins. TOP2β, a target of several chemotherapeutic drugs including VP-16, was abundantly expressed in the GCs of growing follicles. GC-specific deletion of Top2b using Cyp19-Cre caused DNA damage accumulations in these cells, follicle atresia, and decreased ovulation in response to exogenous gonadotropins. The ovaries of Top2b conditional knockout mice were also more sensitive to low-dose genotoxin treatment than wild-type mice ovaries. Thus, our results indicate that GCs are hypersensitive to genotoxic chemotherapeutic drugs and can activate the canonical DNA-damage checkpoint and the p53-dependent apoptotic pathway in response to insults that damage DNA. We also newly identified TOP2β as a factor involved in regulating GC genomic integrity and follicle atresia. This study has clinical implications for ovarian functional defects both for premenopausal cancer survivors and healthy women.