Type II small peptide transporter(PepT2) gene of the Ctenopharyngodon idellus was cloned using RT-PCR and RACE methods, and analyzed using bioinformatics and collinearity methods. The expression of PepT2 gene in different tissues of healthy Ctenopharyngodon idellus was detected and analyzed by real-time fluorescence quantitative PCR. The results showed that the full-length cDNA sequence of PepT2 had 2733 bp, including 2169 bp as an open reading frame encoding a 722-amino-acid peptide, 82 bp at 5′UTR and 482 bp at 3′UTR. PepT2 gene of Ctenopharyngodon idellus contained 20 exons and 19 introns. The predicted relative molecular mass was 8.032×104 and pI at 6.01. The protein had 12 helical transmembrane structures similar to the mammalian homologous proteins, and there was a large outer ring between the transmembrane regions 9 and 10. The transmembrane amino acid region was highly conserved, and contained 6 protein kinase C phosphorylation sites and 3 N-glycosylation sites. The tertiary structure of PepT2 protein was predicted to contain 18 α-helices and 21 β-folds. The amino acid sequence homology analysis results showed that the PepT2 amino acid sequence of Ctenopharyngodon idellus had the highest homology with Danio rerio(86.65%), and the homology with other species were 52.83%-68.15%. Phylogenetic tree analysis showed that Ctenopharyngodon idellus and Danio rerio were the most closely related. Compared with Danio rerio, most genes were retained in the PepT2 chromosome of Ctenopharyngodon idellus with conserved gene blocks. Real-time fluorescence quantitative expression analysis showed that the PepT2 expression level of Ctenopharyngodon idellus was the highest in the intestinal tract, followed by the kidney.