The present study was conducted to estimate the quantitative requirement of L-isoleucine in Japanese seabass(Lateolabrax japonicus)[initial body weight(159.33±1.20)g]by feeding six isonitrogenous and isoenergetic practical diets containing graded levels of L-isoleucine 0.72%, 1.11%, 1.53%, 1.93%, 2.31% and 2.72%(dry weight), respectively, named diet 1—diet 6.After a 10-week growth trial, results showed that weight gain rate(WGR), feed efficiency(FE)and protein retention(PR)were significantly influenced by the different diets(P<0.05).The values increased first, and then declined as the dietary L-isoleucine levels increased.The highest values of these three parameters were 108.55%, 0.89 and 37.57% in fish fed the diet with 1.93% dry matter of dietary L-isoleucine, respectively.Survival rate(SR), feed intake(FI), hepatosomatic index(HSI), viscerosomatic index(VSI), condition factor(CF), body composition and the essential amino acids of muscle were not significantly influenced by dietary L-isoleucine level(P>0.05).Dietary L-isoleucine significantly affected the activities of glutamic-oxaloacetic transaminase(GOT)and glutamic-pyruvic transaminase(GPT)in livers(P<0.05).The level of triglicerid(TG)in serum was significantly affected by dietary L-isoleucine levels(P<0.05).Fish fed the diet with 1.93% L-isoleucine had the highest TG value.GOT and GPT activities and total cholesterol(TC)levels in serum were not significantly affected(P>0.05).In conclusion, appropriate dietary L-isoleucine level can improve the growth of the seabass.On the basis of WGR and PR, using the second-order regression analysis, dietary L-isoleucine requirement of Japanese seabass in the middle growing stage were estimated to be 1.88% and 1.84% dry diet(4.41% and 4.32% dietary protein), respectively.