Mycobacteriophages　express　various　peptides/proteins　to　infect　Mycobacterium　tuberculosis　(M.　tb).　Particular　attention　has　been　paid　to　mycobacteriophage-derived　endolysin　proteins.　We　herein　characterized　a　small　mycobacteriophage-derived　peptide　designated　AK15　with　potent　anti-M.　tb　activity.　AK15　adopted　cationic　amphiphilic　α-helical　structure,　and　on　the　basis　of　this　structure,　we　designed　six　isomers　with　increased　hydrophobic　moment　by　rearranging　amino　acid　residues　of　the　helix.　We　found　that　one　of　these　isomers,　AK15-6,　exhibits　enhanced　anti-mycobacterial　efficiency.　Both　AK15　and　AK15-6　directly　inhibited　M.　tb　by　trehalose　6,6＇-dimycolate　(TDM)　binding　and　membrane　disruption.　They　both　exhibited　bactericidal　activity,　cell　selectivity,　and　synergistic　effects　with　rifampicin,　and　neither　induced　drug　resistance　to　M.　tb.　They　efficiently　attenuated　mycobacterial　load　in　the　lungs　of　M.　tb-infected　mice.　We　observed　that　lysine,　arginine,　tryptophan,　and　an　α-helix　are　key　structural　requirements　for　their　direct　anti-mycobacterial　action.　Of　note,　they　also　exhibited　immunomodulatory　effects,　including　inhibition　of　proinflammatory　response　in　TDM-stimulated　or　M.　tb-infected　murine　bone　marrow-derived　macrophages　(BMDMs)　and　M.tb-infected　mice　and　induction　of　only　a　modest　level　of　cytokine　(tumor　necrosis　factorα　(TNF-α)　and　interleukin-6　(IL-6))　production　in　murine　BMDMs　and　a　T-cell　cytokine　(interferin-　γ　(IFN-γ)　and　TNF-α)　response　in　murine　lung　and　spleen.　In　summary,　characterization　of　a　small　mycobacteriophage-　derived　peptide　and　its　improved　isomer　revealed　that　both　efficiently　restrain　M.　tb　infection　via　dual　mycobactericidal-　immunoregulatory　activities.　Our　work　provides　clues　for　identifying　small　mycobacteriophage-derived　anti-mycobacterial　peptides　and　improving　those　that　have　cationic　amphiphilic　α-helices.　©　2019　Yang　et　al.　Published　under　exclusive　license　by　The　American　Society　for　Biochemistry　and　Molecular　Biology,　Inc.