ABSTRACT: Effects of probiotics on growth, stress tolerance and non-specific immune response in Japanese flounder Paralichthys olivaceus were evaluated in a closed recirculating system. Survival and growth of flounder treated by supplying commercial probiotics either in the diet (the probiotic diet group), or into the rearing water (the water supply group), were higher compared to the untreated group (the control group). Water quality parameters, pH, NH4-N, NO2-N and PO4-P showed lower concentration in the probiotic diet group compared with the control group and the supply group. Plasma lysozyme activity in the probiotic diet group and the water supply group was significantly higher (P < 0.05) than that in the control group. In heat shock stress tests, flounder in the probiotics-treated groups showed greater heat tolerance (measured by 50% lethal time, LT50) than the control group. Pathogen challenge tests with Vibrio anguillarum (2 × 107 c.f.u./mL) resulted in significantly higher survival in the probiotics-treated groups than the control group. Results indicated that probiotics supplied in the rearing water and the diet of fish enhanced the stress tolerance and the non-specific immune system of Japanese flounder, providing them a higher resistance against stress conditions and pathogens.