Trichokonin Ⅵ, a peptaibol-like antimicrobial peptides isolated from the cultured substrates of trichoderma koningii SMF2, has 20 amino acid residues. The conformational flexibility of trichokonin Ⅵ in organic solvents with different polarities, aqueous solvents and membrane mimic solvents was studied by circular dichroism spectroscopy. Trichokonin Ⅵ takes on a typical α-helical structure in different organic solvents, but helicity decreases in aqueous solvent. The helical content increases with increasing the concentration of TFE up to 30%. In phosphate buffered saline, the CD spectrum of trichokonin Ⅵ is concentration dependent, and the intensity of the peaks increases with increasing the concentration of trichokonin Ⅵ. SDS induces a significant transition towards a helix formation, and the CD spectra in membrane mimic solvents increase helicity compared with those recorded without membrane mimic solvents, suggesting the interaction of the peptides with the membrane.