Besides our previous experimental discovery(Zhao Y R, et al. 2015 Langmuir, 31, 12975) that acetonitrile(ACN)can tune the morphological features of nanostructures self-assembled by short peptides KIIIIK(KI4K) in aqueous solution,further experiments reported in this work demonstrate that ACN can also tune the mass of the self-assembled nanostructures.To understand the microscopic mechanism how ACN molecules interfere peptide self-assembly process, we conducted a series of molecular dynamics simulations on a monomer, a cross-β sheet structure, and a proto-fibril of KI4 K in pure water, pure ACN, and ACN-water mixtures, respectively. The simulation results indicate that ACN enhances the intra-sheet interaction dominated by the hydrogen bonding(H-bonding) interactions between peptide backbones, but weakens the inter-sheet interaction dominated by the interactions between hydrophobic side chains. Through analyzing the correlations between different groups of solvent and peptides and the solvent behaviors around the proto-fibril, we have found that both the polar and nonpolar groups of ACN play significant roles in causing the opposite effects on intermolecular interactions among peptides. The weaker correlation of the polar group of ACN than water molecule with the peptide backbone enhances H-bonding interactions between peptides in the proto-fibril. The stronger correlation of the nonpolar group of ACN than water molecule with the peptide side chain leads to the accumulation of ACN molecules around the proto-fibril with their hydrophilic groups exposed to water, which in turn allows more water molecules close to the proto-fibril surface and weakens the inter-sheet interactions. The two opposite effects caused by ACN form a microscopic mechanism clearly explaining our experimental observations. That acetonitrile (ACN) can tune the morphological features of nanostructures self-assembled by short peptides KIIIIK (KI4K) in aqueous solution, further experiments reported in this work demonstrate that ACN can also tune the mass of the self-assembled nanostructures. To understand the microscopic mechanism how ACN molecules are peptide self-assembly processes, we conducted a series of molecular dynamics simulations on a monomer, a cross-β sheet structure, and a proto-fibril of KI4 K in pure water, pure ACN, and ACN-water mixtures, respectively. The simulation results indicate that ACN enhances the intra-sheet interaction dominated by the hydrogen bonding (H-bonding) interactions between peptide backbones, but weakens the inter-sheet interaction dominated by the interactions between hydrophobic side chains. Through the correlations between different groups of solvent and peptides and the so lvent behaviors around the proto-fibril, we have found that both the polar and nonpolar groups of ACN play significant roles in causing the opposite effects on intermolecular interactions among peptides. The weaker correlation of the polar group of ACN than water molecules with the peptide backbone enhances H-bonding interactions between peptides in the proto-fibril. The stronger correlation of the nonpolar group of ACN than water molecules with the peptide side chain leads to the accumulation of ACN molecules around the proto-fibril with their hydrophilic groups exposed to water, which in turn allows more water molecules close to the proto-fibril surface and weakens the inter-sheet interactions. The two opposite effects caused by ACN form a microscopic mechanism clearly explaining our experimental observations.