Aim: To assess Pediococcus acidilactici as a dietary supplement for on-growing red tilapia (Oreochromis niloticus). Methods and Results: Tilapia were fed either a control diet or control diet supplemented with Ped. acidilactici at 107 CFU g-1 for 32 days. Ped. acidilactici colonized the intestinal tract and significantly affected the intestinal microbial communities. PCR-DGGE revealed direct antagonism of gastric Ped. acidilactici with an endogenous uncultured bacterium during a period of reverting to nonsupplemented feeding. Light microscopy revealed that gut integrity and leucocyte levels were unaffected by Ped. acidilactici; however, blood leucocyte levels and serum lysozyme activity were elevated after 14-days' feeding. No significant improvements in growth performance were observed at the end of the trial (day 32), but survival was significantly higher in the probiotic group. Conclusions: The study demonstrates that oral supplementation of Ped. acidilactici modulates intestinal bacterial communities in on-growing red tilapia and also stimulates some aspects of the nonspecific immune response. Significance and Impact of the study: To our knowledge this is the first study assessing the effects of probiotics on the gut microbiota of tilapia using culture-independent methods. Such methods are crucial to understand the mechanisms which underpin and mediate host benefits.