Abstract: The crayfish (Procambarus clarkii) is one of the important economic shrimp species which is widely distributed in the southeastern and central regions of China. In recent years, with the continous rise of peoples’ demand and the continous development of aquaculture industry, various diseases in the aquaculture have frequently outbroken, including bacterial and viral diseases, causing huge economic losses to the crayfish culture industry. As an invertebrate Procambarus clarkii lacks an adaptive immune system, which mainly resists the invasion of pathogens through the innate immune system. Antimicrobial peptide (AMP), as an important effector in the invertebrate’s innate immune system, is one of the substitutes for traditional antibiotics due to its broad-spectrum antibacterial activity and resistance to drug resistance. Crustin is one of the most widely studied antibacterial peptide in Procambarus clarikii. This study amplified and identified a new type of Crustin from Procambarus clarikii, named Pc-CruL. In order to reveal the function of Pc-CruL in Procambarus clarikii, antibacterial activity was determined by means of recombinant expression in vitro. It was hoped to reveal its relationship with pathogens in the form of the prevention and control of the disease and the opening of medicines for Procambarus clarikii.　　Amplifying the Pc-CruL gene in Procambarus clarikii and ligating this gene to the PGEX-4T-1 vector to construct a prokaryotic expression plasmid. The recombinant plasmid was induced and purified in competent BL21 (DE3) for in vitro and in vivo antibacterial experiment. The CDS region of Pc-CruL contains 330 bp and encodes 109 amino acids. Pc-CruL is expressed in all tissues of normal Procambarus clarikii, with higher expression levels in hemocytes and relatively lower expression levels in hepatopancreas. The results of liquid antibacterial assay showed that Pc-CruL protein significantly restricted the growth of Vibrio parahaemolyticus, Staphylococcus aureus, Aeromonas hydrophila, and Aeromonas veronii, and had broad-spectrum antibacterial activity. The injection of exogenous recombinant Pc-CruL protein could improve the survival rate of infection with Vibrio parahaemolyticus. In general, Pc-CruL is an important immune molecule in Procambarus clarikii and plays a vital role in defending against bacterial infections.