Abstract—G(XXKK)nX-CONH2 series antimicrobial peptides have broad-spectral bactericidal activity, high killing rate. The antimicrobial sensitive are affected by many factors. In this article, three cationic amphiphilic peptides G(XXKK)3X-CONH2 (X=I, L and V) have been investigated in pure water and different concentration of 2,2,2-Trifluoroethanol (TFE) by molecular dynamics simulation. The simulations show that the helix conformation becomes more stable as the concentration of TFE increases. Using Leucine (Leu) to replace isoLeucine (Ile) does keep more alpha-helix secondary structure content at low TFE concentration, whereas there is no much difference in helical content of these two peptides when TFE is higher than 30%. The alpha-helix of Valine (Val) mutant is unstable in all TFE concentrations. Instead, Val mutant exhibits a 310-helix conformation at 50% TFE. These simulation results suggest that the stability of alpha-helix is dependent not only on hydrophobic effect but also geometric steric matching and stabilization of TFE. It is found that C-terminus of these peptides are unstable and easy to unfold, which suggests that controlling the unfolding at the C-terminus might be an important strategy to tune the helix stability of these peptides.