To assess Pediococcus acidilactici as a dietary supplement for on-growing red tilapia (Oreochromis niloticus). 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. 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. 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.