Simple SummarySuccessful development of replacement gilts plays a critical role for sustainable swine production, whereas most gilts entering the breeding herd are culled at a young age. Recent advances in reproductive physiology revealed that the ovarian reserve could be considered as an important target of improving reproductive span. Therefore, in the present study, we hypothesized that ovarian follicle development and survival could be enhanced by dietary fiber. Currently there is no appropriate level of dietary fiber intake for growing replacement gilts despite the fact that dietary fiber, rather than starch, protein, or feeding allowance level, could improve the fertility of gilts. Results in the present study demonstrated that ovarian follicle development and survival were sensitive to dietary fiber levels. Our results shed light on the mechanisms underlying the beneficial effects of dietary fiber on the reproduction of sows and provided nutritional insights for enhancing the lifetime fertility of pigs by targeting the ovarian reserve.Ovarian follicle activation and survival were recently found to be controlled by nutrient sensors AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) and apoptosis related markers Caspase-3, Bax, and Bcl-2, yet their expression as regulated by dietary fiber remained uncertain for gilts. To investigate the effects of dietary fiber levels on ovarian follicle development, and the cellular molecular components related to follicle activation and survival of gilts, 76 gilts with similar bodyweight and age were fed four diets, including a corn-soybean meal based control diet, or other three diets to consume 50%, 75%, and 100% more dietary fiber than the control gilts at different experimental phases. Inulin and cellulose (1:4) were added to the corn-soybean meal basal diet to increase dietary fiber content. The growth traits, and the age, bodyweight, and backfat thickness at puberty were not affected by diets. The number of primordial follicles and total follicles per cubic centimeter of ovarian tissue linearly increased with dietary fiber level at day 30 of the experiment and at the 19th day of the 3rd estrous cycle, without negatively affecting the formation of antral follicle with diameter between 1–3 mm or larger than 3 mm. These changes were associated with altered phosphorylation of mTOR, S6, Extracellular regulated protein kinases 1/2 (ERK1/2) and AMPK, and mRNA expression of Caspase-3, Bax, and Bcl-2 in ovarian tissues. Collectively, this study demonstrated a beneficial effect of dietary fiber on the ovarian follicle reserve in gilts, which provides a basis for enhancing reproduction in the short- or long-term.