The three-dimensional structure of Cocaine Amphetamine Transcription Peptide(CART) and the candidate receptor ZMPSTE24 in bovine follicular granulosa cells(GCs) were predicted using homologous modeling. The binding mode between these two molecules was analyzed through Macromolecular docking technology to explore their molecular interactions. Follicular GCs were isolated from three healthy adult cows and transfected with silenced sequences for TEDDM1, CMKLR1, AGTR2 and ZMPSTE24. Total RNA was extracted, and qRT-PCR was employed to assess the relative mRNA expression after silencing the four candidate receptors(TEDDM1, AGTR2, CMKLR1 and ZMPSTE24). The proliferation of GCs in both experimental and control groups was evaluated using the CCK-8 method. Additionally, the mass concentration of estrogen(E2) in the culture medium of each group was determined using the ELISA method, aiming to investigate the functions of these four candidate receptors in bovine follicular GCs. The results revealed one binding site, nine salt bridges, and seventeen hydrogen bonds between ZMPSTE24 and CART. The relative mRNA expression levels of the four candidate receptor test groups were significantly(P<0.01) lower than those of the siNC group and blank group, confirming effective silencing of TEDDM1, CMKLR1, AGTR2 and ZMPSTE24 in GCs. Following the silencing of TEDDM1, CMKLR1, AGTR2 and ZMPSTE24, the cell proliferation rate and E2 mass concentration in the culture medium of each experimental group were significantly(P<0.01) reduced compared to the positive control group(without CART). These findings indicated that even after the silencing of the four candidate receptor genes, CART continues to exert inhibitory effects on GCs proliferation and E2 secretion.