Our recent sum frequency generation (SFG) vibrational spectroscopic experiment ( J. Phys. Chem. B 2014 , 118 , 2904 - 2912 ) showed that immobilized antimicrobial peptide cecropin P1 (cCP1) on a self-assembled monolayer (SAM) surface via N-terminus exhibited significantly different conformational and/or orientational behaviors when exposed to pure water vs a 50% (v/v) 2,2,2-trifluoroethanol (TFE)/water mixture. Meanwhile, our recent molecular dynamics (MD) simulations ( J. Phys. Chem. B 2014 , 118 , 5670 - 5680 ) further revealed that the immobilized cCP1 via N-terminus in pure water largely adopts an overall bent structure lying down on the SAM surface, consistent with the SFG observation. Here, MD simulations were performed on the immobilized cCP1 on a SAM surface via N-terminus while in contact with a 50% (v/v) TFE/water mixture to further investigate the effects of environment (water vs TFE/water mixture) on the interfacial structure and orientation of immobilized peptide. The simulation results demonstrated that the immobilized cCP1 on the SAM surface via the N-terminus with two different starting states with different orientations and conformations, when exposed to a 50% (v/v) TFE/water mixture, was eventually able to maintain a linear a-helical structure, standing upright on the SAM surface. Taken with the corresponding SFG observation, our simulation results indicate that the conformational behavior of the immobilized peptide is mediated by the local hydrophobic environments resulting from the TFE aggregation around the peptide. Such knowledge can be used to regulate the surface conformation and functionality of immobilized peptides via changing surrounding chemical environments (e.g., TFE cosolvent), which is important for the microbial detection and killing based on surface-immobilized antimicrobial peptides.