Penaeidins were important immunity effector molecules, which played a crucial role in innate immunity system of penaeid shrimp. Here, we reported two penaeidin isoforms from Litopenaeus vannamei, which were named as Lva-PEN 2 and Lva-PEN 3 according to the respective structure features. The results of amino acid sequence multiple alignments showed that high similarities existed among these penaeidins. A signal peptide sequence and a cysteine-rich domain which contained six cysteine amino acid residues existed in each penaeidin at the same time. Besides, there was a proline-rich motif which was composed of eight proline residues existed in each penaeidin. After the expression profile analysis was studied, we found that both Lva-pen 2 and Lva-pen 3 transcripts were up-regulated at 48 h after bacteria challenge. Except for the antibacterial activity against Gram-positive bacteria like other penaeidins, these two penaeidins could strongly bind to bacteria and possessed antiproteinase activity. It was speculated that these two penaeidins could prevent bacteria pervasion through bacteria-binding activity and antiproteinase activity. Meantime, the proline-rich motif played a crucial role in these biological processes.