Spontaneous Membrane Translocating Peptides (SMTPs) can translocate silently across the bilayer and, thus, have the best potential to improve the delivery of therapeutic molecules to cells without toxicity. However, how their translocation mechanisms are affected by a specific peptide sequence remains poorly understood. Here, bias-exchange metadynamics simulations were employed to investigate the translocation mechanisms of five SMTPs with the same composition of amino acids (LLRLR, LRLLR, LLLRR, RLLLR, and LRLRL). Simulation results yield sequence-dependent free energy barrier using the FESs along the z-directional distance. An in-depth analysis of sequence-dependent interactions in different regions of the bilayers indicates that the free-energy barrier height of a specific sequence is resulted from the accessibility balance of isolated or clustered hydrophobic residues (L) and hydrophilic residues (R) that leads to different levels of resistance for moving of a peptide into the hydrophobic center of the membrane. At the maximal of the free-energy barrier, all peptides have a conformation parallel to the membrane surface with the barrier height determined by their affinity to the hydrophobic region. The appropriate bilayer perturbation and GDM pairing are beneficial for peptide translocation. These results provide an improved microscopic understanding of how peptide sequence influences the translocation efficiency and mechanism.