Choline is an essential nutrient that has been shown to play critical roles in the development and function of organs such as the brain and liver. It is reported that aberrant choline metabolism occurs in many ovarian-related diseases. However, the specific role of choline in ovarian development and its underlying mechanism is not well understood. To evaluate the in vivo effect of choline supplements on porcine ovarian development, gilts were fed with choline supplement feed from the age of 90 days to 187 days, and their reproductive development were assessed after euthanization. Our results revealed that choline additive increased the number of corpus luteum in the ovary as well as increased the vagina-cervix-length. RT-qPCR results showed that choline supplement regulates the steroid biosynthesis-related genes expression and circulating level of ovarian development-related miRNAs. Moreover, choline increased the alpha diversity of the gut microbiome and enriched the pathways of the gut microbiota that are associated with steroid hormone biosynthesis and infertility-causing environmental pollutant degradation. In addition, choline also regulates the proliferation, apoptosis and innate immunity-related gene expression of the intestinal cells in vitro. In summary, this study demonstrated choline’s influence on ovarian function in vivo. Cumulus-oocyte complex (COC) expansion and oocyte maturation are essential for maintaining ovarian follicular development and female fertility in both human beings and animals. To further investigate the role of miR-29b-3p in the ovarian follicular development, gain- and loss function studies were performed in COC. It was found that over-expression of miR-29b-3p inhibits COC expansion and oocyte maturation in vitro. Further studies revealed that miR-29b-3p induced ROS accumulation by targeting the 3’UTR of CYCS, which ultimately resulted in the suppression of in vitro maturation. In addition, miR-29b-3p over-expression initiate autophagy in granulosa cell by reducing AKT level. I also demonstrated the possible involvement of CYCS/ ROS pathway in this regulating process by the miRNA. In conclusion, this study revealed the role of choline supplementation and miR-29b-3p in porcine ovarian follicular development and their underlying mechanisms, which provides insights into the potential use of choline and miRNAs in improving ovarian function and fertility.