Sperm motilityis akey trait influencing male reproductive performance in poultry. To systematically elucidate the intrinsic regulatory mechanisms governing sperm motility in Yu Fen No.1laying hens, this study employed high- and low-motilityspermsamples from this breed, integrating transcriptomic and proteomic data for multidimensional analysis. The findings revealed significant functional synergistic effects among differentiallyexpressed genes and proteins in energy metabolism and cell motility pathways. Further interaction network analysis identified testicular regulatory genes such as CRH and NPSR1,alongside sperm-specific functional proteins including ENO4 and ASS1,establishing for the first time a hierarchical regulatory model linking testis gene programming to sperm protein execution. This study mechanistically elucidates the decisive role of post-transcriptional fine-tuning in determining sperm motility performance, providing crucial molecular targets and theoretical underpinnings for improving poultry semen quality and advancing breeding technologies