Abstract: To explore the inhibitory effect of myristic acid against suilysin biological activity and the interactive mechanism, pore-forming assay, oligomerization assay, molecular docking, molecular dynamics simulation, minimal inhibitory concentration assay and lactate dehydrogenase activity assays were performed. The results showed that the formation of suilysin oligomer significant decreased when the concentration of myristic acid was 16 μg/mL, thereby inhibiting the pore-forming activity of suilysin, the release of hemoglobin was 8.88% of the control group, and the inhibition rate reached 91.12%. The results of molecular docking and molecular dynamics simulation showed that myristic bound to the junction of domain one, two and three, and 82Asn, 83Asn, 84Ser, 87Ile, 88Ala, 90Ile, 193Phe, 194Gly, 275Phe, 372Ile, 373Leu, 374Ser contributed higher energy which were the critical residues during the binding. Myristic did not showed anti-Streptococcus suis character (MIC values was 128 μg/mL), and the release of lactic dehydrogenase in suilysin or Streptococcus suis treatment group was 67.84% and 45.72% of the control group when the cells received 16 μg/mL myristic treatment. Taken together, myristic inhibited the pore-forming of suilysin by affecting the formation of its oligomer based on a direct binding and protected cells from suilysin or Streptococcus suis cytotoxicity which promised it a candidate for developing anti-Streptococcus suis infection drug.