A novel α-helical antimicrobial peptide G6 rich in Val/Arg residues has been screened previously. In this study, we further evaluated the biochemical stability, interaction with whole bacteria, and in vivo therapeutic or prophylactic role of the peptide in Salmonella typhimurium-infected mice. The results showed that G6 exhibited strong resistance to pH, heat, and salts. But G6 lost the antimicrobial activity when treated with proteolytic enzymes. G6 had no toxicity on mammalian cell. An intraperitoneal model of sepsis caused by Salmonella typhimurium was established in mice. G6 was administered intraperitoneally 1 h before or after mice were infected with Salmonella typhimurium. For the mice given peptide post-bacterial infection, the mortality of the mice and the peritoneal bacterial counts were significantly lower in the groups that were administered 2.5 mg/kg BW and 5.0 mg/kg BW of G6 (P < 0.05) compared to the PBS-treated group. Similar trend was obtained when G6 was given 1 h prior to Salmonella typhimurium infection. Peptide-membrane experiments showed that G6 was effective in permeabilizing the outer and inner membrane in a dose dependent manner, indicating that the peptide targets the cell membrane. Taken together, the results revealed that the peptide G6 may provide a useful alternative to antibiotic agents to treat or prevent bacterial infections.