This study was conducted to determine the optimum highly unsaturated fatty acid (HUFA) level in semi-purified diets for female Chinese mitten crab Eriocheir sinensis broodstock. Five isolipidic and isonitrogenous diets were formulated to contain different HUFA levels (0.14, 0.65, 1.15, 1.64, and 2.07%, respectively) by the supplementation of HUFA-rich fish oil. Diets were fed to triplicate groups of 25 female crabs (75 animals per treatment) stocked in concrete tanks for 8 months. The hepatopancreas, ovary, and muscle of the crabs were sampled after 3-month culturing, and the eggs were sampled after 8-month culturing. The reproductive performance of E. sinensis broodstock was evaluated in relation to the different dietary HUFA levels. Moreover, the fatty acid composition of the hepatopancreas, ovary, muscle, and eggs was determined in crabs fed various levels of HUFA. The results showed that a significant positive correlation was observed between the dietary HUFA level and the HUFA content in hepatopancreas, ovary, and egg, respectively. However, no significant difference in gonadosomatic index, hepatosomatic index, and total ovarian and hepatopancreatic lipid content among the treatments was observed after 3-month culturing. Also, the reproductive performance was similar for all treatments after 8-month culturing. The fact of lacking significant correlation between reproductive parameters and dietary HUFA level was discussed in respect of the catadromous life cycle of E. sinensis and warranted further research with more comprehensive approaches