C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectin gene in different tissues, cellular localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense (MnLec3) was obtained using rapid amplification of cDNA ends method (RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and at different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRT-PCR. The full-length cDNA sequence of MnLec3 was 1 357 bp, which contained a 5' untranslated region of 125 bp, a 3' untranslated region of 206 bp, a 1 026 bp open reading frame (ORF) encoding 341 amino acids. The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain (CRD). Phylogenetic tree analysis stated that Oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawn was artificially challenged with A. hydrophilia, followed by return to control levels at 96 h post-injection, which were similar to MnLec3 protein expression abundance using Western Blot. The results suggested that MnLec3 might be involved in the immune response against bacteria.