Abstract: Objective: To express antimicrobial peptide Molluscidin with high antibacterial activity in recombinant Pichia pastoris. Methods: The optimized nucleotide sequence of Molluscidin were synthesized according to P. pastoris codon usage frequency. The physicochemical properties were analyzed by bioinformatics. The fragment was ligated to pPICZαA vector after digested with EcoR Ⅰ and Not Ⅰ. The recombinant expression vector was transformed into P. pastoris X-33 by electroporation. The recombinant strains were screened with Zeocin and identified by PCR. The recombinant Molluscidin was induced with methanol, and identified by SDS-PAGE and Western blot. The expression conditions were optimized by methanol concentration and culture time. The antibacterial activity was determined by disk diffusion test. Results: The optimized nucleotide sequence was obtained. Bioinformatics analysis showed that the predicted molecular weight was 6521.87 Da, the isoelectric point was 11.28, the estimated half-life in yeast was more than 20 h, and the peptide was classified as stable. The recombinant expression vector pPICZαA-CgMoCo was successfully constructed and transformed into P. pastoris. SDS-PAGE and Western blot demonstrated that one recombinant strain with high-level expression was obtained. The optimal expression conditions were 30 ℃, 250 r/min, 1.0% methanol for 48~72 h. Antimicrobial assay indicated that the culture medium supernatant containing recombinant Molluscidin had antibacterial activity against Gram-negative (i.e., Escherchia coli and Klebsiella pneumoniae) and Gram-positive (i.e., Staphylococcus aureus and Bacillus subtilis) bacteria. Conclusion: One recombinant P. pastoris X-33/pPICZαA-CgMoCo strain with high-level expression and antibacterial activity of recombinant Molluscidin is screened, which lays a foundation for its production and application, and provides a technical approach for the development of antimicrobial peptide from Mollusks.