Abstract: Antibacterial peptides (AMPs) are the main effector molecules of the innate immune system of invertebrates, and their understanding can aid in discovering new drugs and preventing diseases. In the present study, two type Ⅰ Crustin genes were cloned and identified from the mitten crab Eriocheir sinensis, and they were named as Escrus3 and Escrus4 following the nomenclature formed in this field. The cDNA sequence of EsCrus3 was 567 bp, with an Open Reading Frame (ORF) of 351 bp that encode 116 amino acids (aa). EsCrus4 sequence was 743 bp in length with an ORF of 288 bp that encode a putative protein of 95 aa. The domain structure of these two amino acid sequences was further predicted, and they were found to have a N-terminal signal peptide, a C-terminal WAP (Whey acid protein) domain, and a cysteine-rich region (CRR). An evolutionary tree was constructed based on the BLASTP results and the reported Crustin sequences. The results showed that EsCrus3 had the closest evolutionary relationship with Scylla tranquebarica StCrus (AFI56572), while EsCrus4 displayed the closest evolutionary relationship with Portunus trituberculus PtCrus2 (AFU61578). The two molecules were located at different branches with taxonomic significance, suggesting that they might have different biological functions. The tissue distribution showed that both EsCrus3 and EsCrus4 were highly expressed in the ovary with a similar expression level, while the expression level in other tissues was very low or even not detected. When challenged by Staphylococcus aureus or Aeromonas hydrophila, both EsCrus3 and EsCrus4 were up-regulated. EsCrus3 responded faster and significantly up-regulated at 2h after bacterial challenges, while EsCrus4 was significantly up-regulated at 12—24h after bacterial challenges, suggesting that these two genes may play an important role in the antibacterial immune system of the mitten crab. Recombinant proteins EsCrus3 and EsCrus4 were obtained by expression in E. coli system and purification by affinity column. The results of the microbe-binding assay showed that the recombinant proteins EsCrus3 and EsCrus4 exhibited different binding activities to gram-positive bacteria, gram-negative bacteria and fungi, and their binding activities to polysaccharides were significantly different as well. EsCrus3 showed high binding activities to peptidoglycan (PGN) and lipoteichoic acid (LTA) of gram-positive bacteria, and low binding activities to lipopolysaccharide (LPS) of gram-negative bacteria and β-Glucan of fungal; EsCrus4 only has a significant binding activity to PGN. The above research results show that EsCrus3 responds faster to bacterial challenges and has stronger binding abilities to polysaccharide components of pathogens, suggesting that it plays a more important role in the immune defense system of ovary. In addition, the differences in the expression and function of EsCrus3 and EsCrus4 also provide basic data for the mining of new drugs.