We investigated the structure-antimicrobial activity relationship of tachyplesin I (T-I). Even when Lys¹ and Trp² were both deleted from the N-terminal end of T-I, the antimicrobial activity against gram-negative bacteria was not decreased. But as Lys¹ and Trp² were deleted one by one, the antimicrobial activity against gram-positive bacteria and antiviral activity were gradually decreased. Deletion of two disulfide bridges caused a significant decrease in all activities. The circular dichroism (CD) spectra revealed that the analogs containing the two disulfide bidges took a β-sheet structure and that the analogs without the disulfide bridges took a random coil conformation. These results suggest that the β-sheet structure maintained by two disulfide bridges plays an important role in the antimicrobial activity of T-I.