A 12-week feeding trial was conducted to evaluate the effects of multi-strain probiotics (MSP) in a low fish meal (FM) diet on overall performance, gut microbiota, selected non-specific immune responses and antioxidant enzyme activities of olive flounder (Paralichthys olivaceus) juveniles. A total of 225 healthy olive flounders (initial mean body weight, 13.5 ± 0.01 g) were randomly separated into 3 groups of 75 fish, each group having three replicates of 25 fish; first group was fed with a FM-based control diet (Con), 2nd group was fed with a low-FM diet containing a blend of plant and animal protein meals replacing 30% of the FM protein (FM30), and 3rd group was fed with the FM30 diet supplemented with 108-109 CFU kg-1 of the MSP (Pro). With the exception of lipid retention, which was significantly lower in fish fed the FM30 diet compared to the other two treatments, no other statistically significant differences were recorded with respect to any of the other growth and nutrient utilization parameters. Myeloperoxidase and lysozyme activities of fish fed the Pro diet were much higher and significantly different than those of fish fed the FM30 diet. Glutathione peroxidase activity was significantly higher in Pro- than in Con-fed fish, which, in turn, was significantly higher than FM30-fed fish. Expression of immune-related genes including IL-1β, IL-6, and TNF-α was markedly upregulated in livers of the fish fed Pro diet compared to those fed the Con and FM30 diets. Furthermore, supplementation of MSP in FM30 diet enriched the Lactobacillus abundance in the fish gut as well as predictive gene functions in relation to lipid and carbohydrate metabolisms. These data suggested that the MSP could reduce the potential adverse effects of the low-FM diet and might be used as a healthy immunostimulant for olive flounder.