Abstract: To improve the quality of seed and their performance in high rearing density, this study established a sperm selection technique for grass carp (Ctenopharyngodon idella) based on Percoll density gradient centrifugation. Using a 40%/87.5% Percoll density system, sperm was effectively separated into upper and bottom fractions. Compared with uncentrifuged control sperm, the bottom sperm exhibited significantly reduced CMA3 fluorescence intensity, indicating a higher degree of chromatin condensation. In addition, the bottom sperm showed a higher percentage of motile sperm, average path velocity (VAP) and curvilinear velocity (VCL), and achieved higher fertilization rates in artificial reproduction. Under the same rearing condition, offspring derived from bottom sperm showed significantly higher body weight and survival rates at 90 days post-hatching (dph) than those derived from upper and control sperm. We further investigated the effects of stocking density on the growth performance of grass carp at 150 dph. Under low density conditions (5 kg/m³), after 60 days of rearing, offspring from bottom sperm exhibited 25.38% and 4.84% higher weight gain rate and feed conversion efficiency, respectively, than those from control sperm. Under high density conditions (20 kg/m³), these advantages increased to 69.36% and 8.62%, respectively. In low-density conditions, there was no significant difference in serum cortisol concentrations among offspring from the different sperm groups, whereas in high-density conditions, offspring from control sperm exhibited significantly higher serum cortisol concentrations than those from bottom sperm. At last, liver and muscle tissues from offspring derived from bottom, upper, and control sperm under both high- and low-density conditions were subjected to transcriptomic sequencing. Results showed that offspring from bottom sperm had the fewest differentially expressed genes under high-density stress. KEGG enrichment analysis revealed that high-density stress significantly affected pathways related to energy metabolism, immune response, and cellular stress in both liver and muscle tissues. Furthermore, qRT-PCR validation of six growth- and immunity-related differentially expressed genes showed a Pearson correlation coefficient of 0.871, and indicated significant correlation (P<0.001) between log₁₀ fold changes in RNA-seq and qRT-PCR, confirming the reliability of the transcriptomic analysis. Collectively, these results demonstrate that Percoll density gradient centrifugation is an effective method for isolating high-quality sperm in grass carp, thereby improving offspring quality and enhancing resilience under high rearing densities.