Toll-like receptor 5 (TLR5), a member of Toll-like receptors (TLRs) family and is responsible for the bacterial flagellin recognition in vertebrates, play an important role in innate immunity. In the study, two TLR5 genes of grass carp (Ctenopharyngodon idellus), named CiTLR5a and CiTLR5b, were cloned and analyzed. Both CiTLR5a and CiTLR5b are typical TLR proteins, including LRR motif, transmembrane region and TIR domain. The full-length cDNA of CiTLR5a is 3054 bp long, with a 2646 bp open reading frame (ORF), 78 bp 5' untranslated regions (UTR), and 330 bp 3' UTR. The full-length cDNA of CiTLR5b is 3326 bp, with a 2627 bp ORF, 95 bp 5' UTR, and 594 bp 3' UTR. Phylogenetic analysis showed that CiTLR5a and CiTLR5b were closed to the TLR5 of cirrhinus mrigala, cyprinus_carpio, and danio rerio. Subcellular localization indicated that CiTLR5a and CiTLR5b shared similar localization pattern and may locate in the plasma membrane of transfected cells. Real-time quantitative PCR revealed CiTLR5a and CiTLR5b were constitutively expressed in all examined tissues, whereas the highest expressed tissue differed. Following exposure to flagellin and GCRV, CiTLR5a and CiTLR5b were up-regulated significantly. Moreover, the downstream genes of TLR5 signal pathway such as MyD88, NF-κB, IRF7, IL-1β, and TNF-α also up-regulated significantly, whereas the IκB gene was down-regulated, suggesting that CiTLR5a and CiTLR5b involved in response to flagellin stimulation and GCRV infection. The results obtained in the study would provide a new insight for further understand the function of TLR5 in teleost fish.