Follicle development is a crucial step in mammalian reproductive processes, the specific role of Mfn2 in regulating mitochondrial function and endoplasmic reticulum stress in this process is still unclear, this study aimed to investigate the role of Mfn2 in the follicular development of adult sheep. Large, medium, and small follicles were collected, and granulosa cells (GCs) were isolated from large follicles. The expression levels of Mfn2 in different follicles were detected using qRT-PCR and Western blot, and the localization of Mfn2 in follicles was determined through immunofluorescence. Additionally, the expression levels of the mitochondrial autophagy-related protein Pink1, endoplasmic reticulum stress proteins (Grp78, Perk, Chop), and apoptosis-related proteins (Bcl2 and BAX) were detected. Furthermore, siRNAs were transfected into GCs to knock down Mfn2 expression, and changes in intracellular Ca