Several clinical trials have been conducted to evaluate the use of flavopiridol (FP) to treat a variety of cancers, and almost all cancer drugs were found to be associated with toxicity and side effects. It is not clear whether the use of FP will affect the female reproductive system. Granulosa cells, as the important cells that constitute the follicle, play a crucial role in determining the reproductive ability of females. In this study, we investigated whether different concentrations of FP have a toxic effect on the growth of immortalized human ovarian granulosa cells. The results showed that FP had an inhibitory effect on cell proliferation at a level of nanomole concentration. FP reduced cell proliferation and induced apoptosis by inducing mitochondrial dysfunction and oxidative stress, as well as increasing BAX/BCL2 and pCDK1 levels. These results suggest that toxicity to the reproductive system should be considered when FP is used in clinical applications.