Abstract In homothermic animals, toll-like receptors (TLRs) participate in detecting microbial-associated molecular patterns (MAMPs) of probiotics for activation of intestinal mucosal immunity and modulation of gut microbiota. This study evaluated the possible roles of TLRs in fish intestinal mucosal immune response to probiotic Psychrobacter sp. SE6 and microbiota modulation by examining the dominant microbial community and expression of TLR genes and other genes involved in TLR-mediated signaling within the intestine of grouper Epinephelus coioides. The fish were fed for 60days with three different diets: control (without probiotic), diets T1 and T2 supplemented with 1.0×108cellsg−1 viable and heat-inactivated Psychrobacter sp. SE6, respectively. Upregulated expression of TLR2 and TLR5, adaptor MyD88 and cytokines (IL-1β, IL-8 and TGF-β1) was observed in fish fed the viable SE6, while elevated expression of TLR2, but not MyD88 and cytokines, was observed in fish fed the heat-inactivated SE6, which suggested that MyD88-independent TLR2 signaling pathway may be involved in the probiotic recognition in E. coioides. The induced activation of intestinal mucosal immunity, especially the enhanced expression of antibacterial epinecidin-1 and IgM, was consistent with the intestinal microbial data showing that several bacteria were suppressed to undetectable level by both the viable and heat-inactivated SE6. These findings lay the foundation for future studies on the molecular interactions between the probiotics, gut microbiota and mucosal immunity in fish.