Galectins are members of evolutionary conserved lectin family and play important roles in the innate and adaptive immunity of both vertebrates and invertebrates. Galectin-3 is the only chimera galectin with one Cterminal carbohydrate recognition domain (CRD) connected to the N-terminal end. Here, a galectin-3 (named CiGal3) from grass carp was identified and characterized, which encodes polypeptides 362 amino acids with a predicted molecular mass of 36.45 kDa and theoretical isoelectric point of 4.91. The sugar binding motifs involved in carbohydrate binding activity (H-N-R, V-N and W-E-R) were detected in CRD. In comparison to other species, CiGal3 showed the highest similarity and identity to Cyprinus carpio (95.3% sequence similarity and 92.5% sequence identity). The subcellular localization of CiGal3 was distributed in the cytoplasm and nucleus of transfected cells. The CiGal3 transcripts were ubiquitously expressed in all checked tissues and highly expressed in immune tissues. In addition, the expression of CiGal3 in liver and spleen was induced post grass carp reovirus (GCRV), lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C) challenge. These results suggest that CiGal3 plays a vital role in the immune system.