BACKGROUND: Short-chain fatty acids (SCFAs) have emerged as critical modulators of female reproductive function and host gut microbiota. OBJECTIVES: This study aimed to investigate the impact of dietary SCFAs supplementation on reproductive performance and gut microbiota in gilts, and to elucidate the underlying mechanisms. METHODS: Eighty gilts (95 days old) were randomly assigned to either a control group (Ctrl, 40 gilts) receiving a basal diet, or a SCFAs treatment group (SCFAs, 40 gilts) receiving a basal diet supplemented with 0.13% sodium acetate, 0.11% sodium propionate, and 0.09% sodium butyrate. At third estrus, 13 gilts (6 from Ctrl and 7 from SCFAs) were euthanized for follicular development and gut microbiota analysis, while the remaining gilts completed gestation for reproductive performance assessment. RESULTS: SCFAs group had higher total number born (11.97 vs. 9.44) and total number born alive (11.28 vs. 9.34) compared to Ctrl group (P <
0.05). SCFAs group had increased counts of secondary follicles (36.14 vs. 26.83), antral follicles (10.29 vs. 6.67), and corpus luteum (25.09 vs. 19.33), alongside had reduced atretic follicles (15.32 vs. 20.67) compared to Ctrl group (P <
0.05). Proteomic analysis revealed that SCFAs-induced differentially expressed proteins (DEPs) were significantly enriched in the follicular development-related pathways (P <
0.05). Apoptosis-related DEPs positively correlated with follicular development indices (P <
0.05), consistent with the reduced apoptosis observed in ovarian granulosa cells of the SCFAs group. Additionally, SCFAs supplementation improved both the composition and alpha diversity (P <
0.05) of gilts' gut microbiota. Furthermore, both the SCFAs-enriched bacteria and plasma SCFAs concentrations showed positive associations with gilts' follicular development indices (P <
0.05). CONCLUSIONS: Dietary SCFAs supplementation enhances reproductive performance in gilts by promoting ovarian follicular maturation and optimizing gut microbiota composition.