Objective NOBOX is a transcription factor that plays a key role in the activation of primary follicles. It directly regulates the transcription of Gdf9 and participates in the GDF9 / SMAD pathway to affect the proliferation and differentiation of granulosa cells. Kit, an important gene affecting follicular development, has multiple NOBOX binding sites in its promoter. In this study, we aimed to investigate the transcriptional regulation of Kit by the transcription factor NOBOX in the primary follicular development stage as well as the influence of the GDF9 / SMAD pathway on follicular development. Methods The development time of primary follicles in vitro was measured. qRT-PCR was used to detect the mRNA expression changes of Nobox, Kit, Kitl and Gdf9 in primary follicles injected with Nobox-siRNA. Western blot was used to measure protein expression changes for NOBOX, Kit and P-SMAD in primary follicles injected with Nobox-siRNA. ChIP assay was used to identify the binding site of NOBOX on the Kit gene promoter. Results Primary follicles developed into secondary follicles on the 5th day in vitro. Injection of Nobox-siRNA into primary follicles delayed their development into secondary follicles by 2 days. After Nobox-siRNA injection into primary follicles, the expression of Nobox, Kit, Kitl and Gdf9 mRNAs in follicles was significantly downregulated, and the expression levels of NOBOX, Kit and P-SMAD2 / 3 proteins were decreased. NOBOX showed binding to the Kit gene promoter. Conclusions NOBOX is a key gene in the development of primary follicles to secondary follicles in mice. NOBOX directly binds to the Kit gene promoter and affects the KitL/ Kit and GDF9 / SMAD pathways in primary follicles.