The development of pre-recruitment follicles plays a critical role in determining egg-laying performance in poultry. This study combines proteomic and metabolomic analyses to explore changes in proteins and metabolites, to elucidate key regulatory mechanism involved in chicken pre-recruitment follicular development. Histological examination revealed a significant increase in yolk deposition in small yellow follicles (SYF) compared to small white follicles (SWF). Metabolomics analysis identified significantly enriched differential metabolites (DMs) between SWF and SYF in pathways such as Lysosome, Ferroptosis, Biosynthesis of unsaturated fatty acids, and Tryptophan metabolism. Particularly, Adenosine-5'-Diphosphate (ADP) was downregulated during follicular recruitment and was significantly enriched in the lysosome pathway. Proteomic analyses revealed that differentially expressed proteins (DEPs) in SWF and SYF were enriched in pathways including Lysosome, Glutathione metabolism, Cholesterol metabolism, Arginine and proline metabolism, and amino acid biosynthesis. Among these DEPs, NAD-dependent protein deacetylase sirtuin 5 (SIRT5) was significantly upregulated, while lysosomal-associated membrane protein 1 (LAMP1) was down-regulated during the development of pre-recruitment follicles. SIRT5 was linked to the negative regulation of reactive oxygen species metabolism, whereas LAMP1 was associated with lysosome and autophagy pathways. Further validation experiments demonstrated high expression of SIRT5 in SYF, particularly in granulosa cells (GCs). Silencing SIRT5 in GCs resulted in increased ROS production and upregulated expression of autophagy-related proteins LC3Ⅱ and Beclin1, as well as lysosome markers LAMP1. Conversely, lipid droplet deposition and p62 expression were suppressed. inhibited. Taken together, these findings suggest that SIRT5 upregulation promotes the development of pre-recruitment follicles by inhibiting the autophagy-lysosome pathway in chicken GCs.