Semaphorins are a family of evolutionarily conserved morphogenetic molecules that were initially found to be associated with axonal guidance. Semaphorin 4C (Sema4C), a member of the fourth subfamily of semaphorins, has been demonstrated to play multifaceted and important roles in organ development, immune regulation, tumour growth and metastasis. However, it is completely unknown whether Sema4C is involved in the regulation of ovarian function. We found that Sema4C was widely expressed in the stroma, follicles and corpus luteum of mouse ovaries, and its expression was decreased at distinct foci in ovaries of mice of mid-to-advanced reproductive age. Inhibition of Sema4C by the ovarian intrabursal administration of recombinant adeno-associated virus (AAV)-shRNA significantly reduced oestradiol, progesterone and testosterone levels in vivo. Transcriptome sequencing analysis showed changes in pathways related to ovarian steroidogenesis and the actin cytoskeleton. Similarly, knockdown of Sema4C by siRNA interference in mouse primary ovarian granulosa cells (GCs) or thecal interstitial cells (TICs) significantly suppressed ovarian steroidogenesis and led to actin cytoskeleton disorganization. Importantly, the cytoskeleton-related pathway RHOA/ROCK1 was simultaneously inhibited after downregulation of Sema4C. Furthermore, treatment with a ROCK1 agonist after siRNA interference stabilized the actin cytoskeleton and reversed the inhibitory effect on steroid hormones described above. In conclusion, Sema4C may play an important role in ovarian steroidogenesis through regulation of the actin cytoskeleton via the RHOA/ROCK1 signalling pathway. These findings shed new light on the identification of dominant factors involved in the endocrine physiology of female reproduction.© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.