Abstract: The genus Mytilus is not only of significant economic importance in aquaculture around the world but also shows strong tolerance to a wide range of environmental factors, and is, used as a pollution indicator in coastal waters. Due to their filter-feeding habit, Mytilus accumulate large numbers of bacteria from the water. However, Mytilus appear less affected or unharmed by the syndromes and infectious agents that distress other bivalves. The immunity of Mytilus has received increased attention in the last decade, resulting in a valuable increase in the availability of its molecular components for controlling invasive microbes. In which, Mytilus antimicrobial peptides have strong molecular diversity, and therefor become the important object of antimicrobial peptide research. In this study, two novel antimicrobial peptides, myticalin and mytimacin, were identified from the transcriptomic data of M. coruscus hemocytes. However, the molecular features and the immune mechanism of these mussel defensins are unknown. For understanding the relationship of structure-function of myticalin and mytimacin, the two peptides were synthesized by a solid phase polypeptides chemical synthesis technology, and the functions of these two peptides were studied in vivo, including antimicrobial activities against various bacteria and fungus and erythrotoxicity against sheep red blood cell. Further, the mechanism of these two peptides inhibiting microbes were observed using scanning electron microscope. For exploring the immune responses of these two peptides against various bacteria and fungus, the mussel was induced by Staphylococcus aureus, Vibrio alginolyticus and Candida albicans, respectively, and the relative expression level of myticalin and mytimacin were calculated by real-time fluorescent quantitative PCR. The results indicated that, mytimacin of M. coruscus has classical features similar to those of other Mytilus antimicrobial peptides, containing cysteine and disulfide bonds, and thus forming a cysteine stabled α/β structure. While the myticalin is an antimicrobial peptide without cysteine and disulfide bonds, and forms a structure dominated by random coil. Similar changes of surface structure of tested microbes were observed under SEM for both myticalin and mytimacin, in which, agglutination of S. aureus and flake bulge of V. alginolyticus were presented under both myticalin and mytimacin treatment. In addition, holes can be observed for C. albicans after incubation with mytimacin, while no morphological alteration can be observed for C. albicans after incubation with myticalin. Both myticalin and mytimacin showed no erythrotoxicity for sheep red bloods. Moreover, the dynamic changes in the expression level of two peptides after microbial induction were examined, and the results showed a marked increase in relative expression level observed in vivo for both myticalin and mytimacin genes after microbial challenge relative to the respective controls. We noticed that challenges with Gram-positive S. aureus resulted in general up-regulation of myticalin gene expression with more than 7-fold change from 1h to 12h post-induction, indicating a high sensitivity of myticalin to Gram-positive bacteria. Similarly, mytimacin also showed a significant up-relation after S. aureus induction, with a significant up-regulation of gene expression with a more than 2.8-fold change from 2h to 8h post-induction. However, Gram-negative V. alginolyticus only induced a slight up-relation for the expression level of both myticalin and mytimacin genes, with less than 4-fold change and only two time points (2h and 4h for myticalin, and 4h and 8h for mytimacin). These results indicated that both myticalin and mytimacin were relatively insensitive to Gram-negative strains. Interestingly, mytimacin also showed a relative sensitivity to C. albicans, with the high up-regulation can be observed during 2h to 8h post-induction with an up-regulation of more than 3-fold change. Considering that myticalin and mytimacin showed differing sensitivities to different microbes, we might thus speculate that Mytilus would adopt a friendlier attitude towards Gram-negative bacteria, and the weaker mussel AMP response to Gram-negative bacteria may be related to the greater abundance of Gram-negative bacteria in the marine environment. If the mussel responded to all Gram-negative stimuli with high intensity, they could be in danger of high inflammatory stress. In addition, these observations strongly suggested the existence of different recognition mechanisms or signal transduction pathways in mussels for the expression of myticalin and mytimacin. These results provided basis for understanding the molecule mechanism of Mytilus immunology, and the function of novel Mytilus antimicrobial peptides, and thusly provided basis for the molecular resource development of mussel antimicrobial peptides.