Peptide-induced phospholipid flip-flop (scrambling) was evaluated using transmembrane model peptides in which the central residue was substituted with various amino acid residues (sequence: Ac-GKK(LA)nXW(LA)nLKKA-CONH2). Peptides with a strongly hydrophilic residue (X = Q, N, or H) had higher scramblase activity than that of other peptides, and the activity was also dependent on the length of the peptides. Peptides with a hydrophobic stretch of 17 residues showed high flip-promotion propensity, whereas those of 21 and 25 residues did not, suggesting that membrane thinning under negative mismatch conditions promotes the flipping. Interestingly, a hydrophobic stretch of 19 residues intensively promoted phospholipid scrambling and membrane leakage. The distinctive characteristics of the peptide were ascribed by long-term molecular dynamics simulation to the arrangement of central glutamine and terminal four lysine residues on the same side of the helix. The combination of simulated and experimental data enables understanding of the mechanisms by which transmembrane helices, and ultimately unidentified scramblases in biomembranes, cause lipid scrambling.