Toll-like receptor 5 (TLR5) is a member of TLRs family responsible for the bacterial flagellin recognition in vertebrates. Herein, the TLR5M gene structure of Pacific red snapper (Lutjanus peru) was characterized. The full-length cDNA of LpTLR5M comprises an open reading frame (ORF) of 2715 bp, encoding a polypeptide of 904 amino acids including 9 LRRs (residues 119–562) and one LRR-CT domain (residues 593–646) at the extracellular region, and a TIR domain (residues 710–904) in the cytoplasmic region. The amino acid sequence in L. peru TLR5 showed high identity (66–69%) with TLR5 from Paralichthys olivaceus and Scophthalmus maximus. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of LpTLR5M mRNA in all the examined tissues, with higher levels in intestine, liver, and head-kidney. Furthermore, expression of LpTLR5M and five cytokine genes was also investigated 24 h and one week post-stimulation in fish intraperitoneally injected with ToxA, live V. parahaemolyticus (Vp) or V. parahaemolyticus Lysate antigens. TLR5M was significantly induced in fish infected with Vp. The pro-inflammatory cytokines IL-6, IL8 and IL-12 were significantly up-regulated in head-kidney in fish stimulated with Vp, while in intestine upregulation was observed following ToxA or Lysate injection. In contrast, IL-17 mRNA was significantly up-regulated in the intestine from fish infected with live Vp at 24 h post-injection. The results indicate that Lysate and Vp antigens can induce an immune response via TLR5M and that cytokines have an important role in the defense mechanisms against V. parahaemolyticus.