Neddylation is a biological process that covalently links neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) to its substrate. Its main function is to activate the cullin ubiquitin junction complex, thereby promoting substrate protein degradation. The research of neddylation mainly focus on cell senescence, Alzheimer's disease progression, and tumor treatment. However, the neddylation mechanism underlying follicular granulosa cells (GCs) proliferation and steroidogenesis remains unclear. Therefore, this study aimed to reveal the functions of neddylation in sheep follicular GCs. Here, immunohistochemistry results revealed that NEDD8 was expressed in sheep follicular GCs. The neddylation-specific inhibitor MLN4924 and NEDD8 small interfering (si) RNA (si-NEDD8) were used to inhibit neddylation, significantly attenuating sheep follicular GC proliferation. The RNA sequencing (RNA-Seq) showed that cytochrome P450 11A1 (CYP11A1) and steroidogenic acute regulator (StAR) were significantly downregulated via the inhibition of neddylation. Additionally, qRT-PCR results showed that CYP11A1, StAR and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA abundance decreased significantly following the addition of various MLN4924 doses and si-NEDD8, however, CYP19A1 mRNA levels did not significantly differ. Western blotting results showed that inhibition of neddylation also significantly decreased the expression of CYP11A1, 3β-HSD, cytochrome P450 19A1 (CYP19A1), StAR, and androgen receptor (AR). The current study revealed that the inhibition of neddylation affects sheep follicular GC function by inhibiting proliferation and the enzymes involved in synthesis in sheep.