It is well documented that granulosa cell apoptosis is the main reason for follicular atresia and death, however, increasing evidence suggests that autophagy plays an important role in the fate of granulosa cells. MiR-21-3p regulated many fundamental biological processes and was pivotal in autophagy of tumor cell, nevertheless, the autophagy in cattle ovary and how miR-21-3p regulates the follicular cells is unknown. In this study, we aimed to elucidate the autophagy and the role of miR-21-3p in cattle ovary using bovine primary ovarian granulosa cells (BGCs). The results showed the autophagy for the first time in BGCs in large follicle according to autophagic gene transcript of LC3, BECN-1, ATG3, protein expression of LC3, P62 and LC3 puncta, a standard marker for autophagosomes. MiR-21-3p was identified as a novel miRNA that repressed BGCs autophagy according to the results from plasmids transfection of miR-21-3p mimics and inhibitor. Meanwhile, VEGFA was confirmed to be a validated target of miR-21-3p in BGCs using luciferase reporter assays and the results of VEGFA expression decreased with transfection of miR-21-3p mimics, while increased with transfection of miR-21-3p inhibitor. In addition, small interference mediated knockdown of VEGFA significantly inhibits BGCs autophagy signaling, however, overexpression of VEGFA in BGCs promoted autophagy in the presence of miR-21-3p. Finally, the results of AKT and it's phosphorylation suggested that miR-21-3p suppressed VEGFA expression through downregulating AKT phosphorylation signaling. In summary, this study demonstrates that miR-21-3p inhibits BGCs autophagy by targeting VEGFA and attenuating PI3K/AKT signaling.