Background: Female infertility is a health issue for both humans and animals and despite developments in medical interventions, there are still some conditions that cannot be treated successfully. It is important to explore other potential therapies or remedies that could improve reproductive health. Choline is an over-the-counter supplement and essential nutrient that has many health benefits. It has been suggested to be beneficial in various aspects of fertility, including fetal development and endocrine disorders like polycystic ovarian syndrome (PCOS). However, choline’s impact on ovarian function has not been explored. Methods: To study the effects of choline on ovarian development, 36 female Yorkshire \times Landrace pigs were fed the following four supplemented diets between 90 and 186 days of age: (1) Control (corn and soybean meal-based diet that met estimated nutrient requirements, n = 9); (2) Choline (additional 500 mg choline per 1 kg of control diet, n = 8); (3) Omega-3 (additional 5556 mg Omega-3 per 1 kg control diet by introducing fish oil); (4) Choline + Omega-3 (500 mg choline + 5556 mg Omega-3 per 1 kg control diet). Pigs fed the choline-supplemented diet were compared to the control group and those fed diets supplemented with Omega-3 as fertility-promoting agent. Results: It was found that the number of corpus luteum per ovary in the Choline (16.25 \pm 2.88), Omega-3 (10.78 \pm 1.71) and Choline + Omega-3 (14.89 \pm 2.97) groups were all higher in comparison to that of the control group (5.56 \pm 1.72, p 0.05). The percentage of antral follicles in the Choline + Omega-3 group were higher compared to the control group (p 0.05). To elucidate the potential molecular mechanism of choline on these improved ovarian phenotypes, the expression of a group of genes that are involved in ovarian development, including cytochrome P450 family 11 subfamily A member 1 (CYP11A1), follicle stimulating hormone receptor (FHSR) and luteinizing hormone receptor (LHR), was analyzed using RT-qPCR. The expression of both LHR and CYP11A1 was significantly upregulated in the choline-supplemented group (p 0.05), while there are no differences in FSHR expression among all the groups. Additionally, the expression of miR-21, -378, -574, previously found to be important in ovarian function, were examined. Our data showed that miR-574 was upregulated in the Choline group while miR-378 was upregulated in the Choline + Omega-3 group in comparison to the control group (p 0.05). Further, serum metabolite analysis showed that 1-(5Z, 8Z, 11Z, 14Z, 17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, a form of phosphatidylcholine metabolite, was significantly increased in all the treatment groups (p 0.05), while testosterone was significantly increased in both Omega-3 and Choline + Omega-3 groups (p 0.05) and tended to be reduced in the choline-supplemented group (p = 0.08) compared to the control group. Conclusions: Our study demonstrated choline’s influence on ovarian function in vivo, and offered insights into the mechanisms behind its positive effect on ovarian development phenotype.