Antimicrobial peptide F1, a novel antimicrobial peptide from Tibetan kefir, have shown strong antimicrobial activity against several bacteria and fungi. We identified the amino acid sequence and studied the antimicrobial mechanism of peptide F1 against Escherichia coli. Our results showed that antimicrobial peptide F1 contained 18 amino acids (Thr-DAP-Asn-Thr-PEA-His-Pro-Asn-Thr-His-Leu-Ile- PEA-CySH-Val-Asn-PEA-Tau), which increased the outer and inner membrane permeability of E. coli, and the leakage of the cytoplasmic β-galactosidase and potassium ions was detected in the process. Morphologies of E. coli were observed by confocal laser scanning microscopy and transmission electron microscopy, which visually showed that antimicrobial peptide F1 could penetrate and accumulate into cell causing disruption of cell membrane functions. Furthermore, we elucidated the DNA binding ability of antimicrobial peptide F1 by agarose gel retardation and atomic force microscopy. Our findings indicated that antimicrobial peptide F1 has multiple targets in the killing of E. coli.