Abstract: We studied the effects of different feeding frequencies on the food intake, growth, digestive enzymes, antioxidant enzymes and intestinal tissue of Paralichthys olivaceus under the condition of running water culture, in order to explore the optimal feeding frequency of P. olivaceus. The experiment set up three feeding frequency treatment groups, feeding 2 (T2), 3 (T3), 4 (T4) times per day, lasting 60 d. The results show that the daily intake of T3 group was significantly higher than those of T2 and T4 groups (P<0.05), and there was no significant difference in the specific growth rate and survival rate of T2 and T3 groups, which were significantly higher than that of T4 group (Specific growth rate: P<0.05, survival rate: P<0.01). There was significant difference in the weight gain rate among the three groups (P<0.05); there was no significant difference in the hydrogen peroxide enzymes, total superoxide dismutase and lipase between T2 and T3 groups, which were significantly higher than in T4 group (hydrogen peroxide enzyme, lipase: P<0.01; total superoxide dismutase: P<0.05). There was significant difference in gastric proteases among the three groups (P<0.05), with a descending order of T3, T2 and T4. The intestinal wall thickness, longitudinal muscular thickness and number of goblet cell in T4 group were significantly lower than those in T2 and T3 groups (P<0.01), but there was no significant difference between the T2 and T3 groups, and the thickness of the endocrine cells in T3 group was significantly higher than that of the other two groups (P<0.05), and the difference between the T2 and T4 groups was not significant. Combining the results with the actual situation of breeding, under the condition of running water culture, the most suitable feeding frequency in the process of P. olivaceus cultivation is 3 times per day.