This study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) on growth performance, non-specific immunity, expression of some immune-related genes and resistance to Vibrio harveyi in juvenile hybrid grouper (Epinephelus fuscoguttatus ♀×E. lanceolatu♂). Six isoproteic and isolipidic experimental diets were formulated with graded levels of n-3 HUFA (0.65%, 1.00%, 1.35%, 1.70%, 2.05% and 2.40% of dry matter, respectively), and 0.65% group was used as control group. Each diet was randomly allocated to triplicate groups of fish in 1 000 L fiberglass tank, and each tank was stocked with 40 fish [initial weight (12.06 ±0.01) g] for 8 weeks. Results showed that feed conversion ratio (FCR), survival rate (SR), hepatosomatic index (HSI) and condition factor (CF) were not significantly affected by dietary n-3 HUFA levels. Weight gain (WG) and specific growth rate (SGR) in 1.35% group were significantly higher than those in 2.40% group. Crude lipid of body in 1.00% group was significantly lower than those in 1.70% and 2.40% groups Liver and muscle fatty acid profiles reflected that of diets. Before challenge, the activity of serum superoxide dismutase (SOD), catalase (CAT) and content of complement 3 (C3) in 1.35% and 1.70% groups significantly higher than those of control group. After challenge, serum CAT, glutathione peroxidase (GSH-Px), lysozyme (LZM) and C3 all increased sharply, while SOD showed the opposite trend. Before challenge, the expression levels of intestine toll-like receptor 22 (TLR22) and myeloid differentiation factor 88 (MyD88) mRNA in 2.40% group were significantly increased, and the expression levels of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) mRNA in 2.05% group were significantly higher than those in 1.00% and 1.35% groups. In addition, the TLR22 and IL-1β mRNA levels in kidney of 1.70% group were significantly lower than those in control group. After challenge, the expression level of MyD88 mRNA in intestine of 1.35% group was significantly higher than that in 1.00% group and from 1.70% to 2.40% group, while TNF-α and IL-1β obtained minimum values in 1.70% group. In the kidney, the interleukin 10 (IL10) mRNA expression was significantly higher in 1.70% group than those in other groups, while the IL-1β expression in 1.70% group showed the opposite trend and significantly lower than that in 2.40% group. Results of this study suggested that moderate dietary n-3 HUFA (1.47%-1.70% HUFA) could improve the growth performance, non-specific immunity and inhibit the inflammatory response of hybrid grouper.