[Objective] Listeria monocytogenes, an important food-borne pathogenic bacteria, often causes human and animal diseases. The cell wall surface proteins with an LPXTG motif play an important role in the pathogenic process of L. monocytogenes. According to the whole sequence of the reference strain, the function of some proteins among the 41 predicted LPXTG motif proteins is unknown. The aim of this study is to clone and bioinformatics analysis of the gene of LPXTG motif protein Lmo0160 of L. monocytogenes LM90SB2 isolated from diseased sheep in Xinjiang. It could provide the foundation for functional verification. [Methods] A pair of specific primers was designed according to the predicted sequence of L. monocytogenes lmo0160 gene in GenBank. The lmo0160 gene was amplified by PCR, and the amplified products were cloned into pMD19-T vector and verified by PCR, restriction endonuclease digestion and sequencing. In addition, the bioinformatics on nucleotide sequence of lmo0160 gene of LM90SB2 strain as well as putative protein structure had been analyzed. [Results] The sequence length of LM90SB2 lmo0160 gene was 1 708 bp, containing a 1 428 bp open reading frame (ORF) encoding 475 amino acids. The nucleotide sequence similarity of lmo0160 gene of LM90SB2 strain was 99.0%?99.1% with FSAN008100 strain (serotype 4b, USA), CFSAN023463 strain (serotype 4b, USA), J2-064 strain (serotype 4b, USA), F2365 strain (serotype 4b, cheese isolate, USA) and NTSN strain (serotype 4b, sheep brain isolate, Yangzhou, China), 97.2% with M7 strain (serotype 4a, cow’s milk isolated, Zhejiang, China), and 87.2%?91.1% with Finland 1998 strain (serotype 1/2a, USA), N53-1 strain (serotype 1/2a, fish isolate, Denmark), N1546 strain (serotype 1/2a, cooked ham isolate, Switzerland) and EGD-e strain (serotype 1/2a, USA). Its putative amino acid sequence similarity was 91.8%?99.4% with above strains. The phylogenetic tree showed that the lmo0160 gene of LM90SB2 strain was closely relative with CFSAN023463, F2365 and NTSN strains which were in the same branch. However, it was genetically far to the international standard strain EGD-e. The secondary structure prediction showed that LM90SB2 Lmo0160 was hydrophilic protein without signal peptide and transmembrane structure. The protein domain prediction showed that Lmo0160 protein had collagen binding domain and collagen-binding surface protein Cna-like, B-type domain (Cna B). [Conclusion] The lmo0160 gene of LM90SB2 strain was successfully cloned. The cloning and analysis of lmo0160 gene provide an important foundation for further study of lmo0160 gene.