Abstract: A study was conducted to evaluate the effects of different supplemental methods of phytase on growth and feed utilization of Japanese flounder (Paralichthys olivaceus). The phytase-pretreated soybean meal (PP-SBM) was obtained by fermentation with Candida utilis. Four isonitrogenous (49.7% crude protein) and isocaloric (20.9 kJ/g) diets were formulated. The control diet contained 68.0% fish meal (FM) as the main protein source. In soybean meal (SBM) diets with 0 and 1000 IU/kg phytase, 45% of FM protein was replaced by SBM protein. The PP-SBM diet was formulated in which phytase-pretreated soybean meal protein replaced 45% of FM protein. Each diet was randomly fed to triplicate groups of Japanese flounder with initial weight (2.02 ± 0.02) g/fish. Fish were fed to apparent satiation twice daily (08:00 and 16:00). During the experiment, water temperature fluctuated from 22.5 to 25.5℃, salinity from 29.5 to 32.0 and dissolved oxygen was not less than 7.0 mg/L. At the end of the 10-week feeding trial, survival and feed intake showed no significant difference among dietary treatments (P>0.05). The specific growth rate (SGR), feed efficiency ratio (FER), protein efficiency ratio (PER), nitrogen retention (P0.05). The SGR, FER, PER and nitrogen retention showed no significant difference among fish fed the SBM diet supplemented phytase, PP-SBM diet and control diet (P>0.05). Comparing with the SBM diet without phytase, the fish fed the SBM diet supplemented phytase showed significantly higher SGR (P0.05). The content of phytate in phytase- pretreated SBM was lower than that in SBM. The fish fed the diet contained phytase-pretreated SBM exhibited significantly higher SGR (P0.05). The whole-body crude protein (P0.05). The ash in fish fed the SBM diet without phytase, SBM diet supplemented phytase and PP-SBM diet was significantly higher than that in fish fed FM diet (P<0.05). The results indicate that supplementation of phytase to the SBM diet can improve growth response and utilization of nitrogen and phosphorus, and the pretreatment of soybean meal with 1000 IU/kg diet phytase can improve the growth responses and feed utilization of Japanese flounder (Paralichthys olivaceus).