The effects of different dietary nucleotide mixture (NT) levels (0, 0.2, 0.4 and 0.6%) was investigated on thereproductive performance, fatty acid profile and biochemical parametersin Litopenaeus vannamei female that were co-fedwith fresh food (2 times daily) and experimental diets (2 times daily) for 30 days. Sampling was carried out at the first day of the trial, before eyestalk ablation (ESA)(day 21) and after ESA (day 30). Reproductive performance parameters including the hepatopancreatic index, absolute fecundity, egg diameter and latency period were higher in the NT supplemented groups than the control group (P < 0.05). Total n–3 polyunsaturated fatty acids in the ovaries, especially eicosapentaenoic acid, were higher in the NT supplemented groups than in the control (222.6±7.3vs. 204.7±3.0 mg g-1 extracted lipid) (P < 0.05).Total hemocyte count, granular cells, plasma glucose (Glu), total protein (TP), calcium (Ca2 ), total cholesterol (Chol), triglyceride (Tg) and high density lipoprotein (HDL)levels were significantlyincreased in theNT supplemented groups in comparison with the control group (P< 0.05). After ESA, the concentration of plasmaGlu, TP, Chol and Tg significantly decreased, but HDL levels increased in all groups (P< 0.05). These results suggested that the application of dietary NT may present a novel strategy for increasing reproductive performance and health status in L. vannamei.