Bovine　lactoferrin　peptide　(LFcinB),　as　an　antimicrobial　peptide,　is　expected　to　be　an　alternative　of　antibiotics　owing　to　its　broad-spectrum　antimicrobial　activity　and　specific　mechanism.　However,　the　weak　antimicrobial　activity,　high　hemolysis,　and　poor　stability　of　LFcinB　limited　its　applications　in　the　field　of　biomedicine,　food　and　agriculture.　In　order　to　improve　the　antimicrobial　activity　of　LFcinB,　five　mutants　were　designed　rationally,　of　which　mutant　LF4　(M10W/P16R/A24L)　showed　highest　antimicrobial　activity.　The　bioinformatics　analysis　indicated　that　the　improved　antimicrobial　activity　of　LF4　was　related　to　its　increased　cations,　higher　amphiphilicity　and　the　extension　of　the　β-sheet　in　the　structure.　These　studies　will　highlight　the　important　role　of　bioinformatic　tools　in　designing　ideal　biopeptides　and　lay　a　foundation　for　further　development　of　antimicrobial　peptides.　©　2023,　The　Author(s),　under　exclusive　licence　to　Springer　Science+Business　Media,　LLC,　part　of　Springer　Nature.