The purpose of this study was to investigate the effects of antimicrobial peptide microcin J25 (MccJ25) on growth performance, immune regulation, and intestinal microbiota in broilers. A total of 3120 one-day-old male Arbor Acres (AA) broilers were randomly allocated to five groups (12 replicates, 52 chickens per replicate). The treatments were control, challenge (0 mg/kg MccJ25), different dosages of antimicrobial peptide (AMP) (0.5 and 1mg/kg MccJ25), and antibiotic groups (20 mg/kg colistin sulfate). The MccJ25 groups increased the body weight gain (starter and overall) that was reduced in the challenge group. The overall (day 1 to day 42) feed-to-gain ratio (G:F) was significantly decreased in AMP groups compared with the challenge group. Birds fed AMP had a decreased population of total anaerobic bacteria (day 21 and day 42) and E. coli (day 21 and day 42) in feces, as well as a lower Salmonella infection rate (day 21 and day 42) compared with birds in the challenge group. The villus height of the duodenum, jejunum, and ileum, as well as the villus height/crypt depth of the duodenum and jejunum were greater in AMP groups than birds in the challenge group. Moreover, MccJ25 linearly improved the villus height of the duodenum and jejunum. The addition of MccJ25 decreased the concentration of TNF-α, IL-1β, and IL-6 compared with challenge group. At d 21, MccJ25 linearly reduced the level of IL-6. In conclusion, dietary supplemented MccJ25 effectively improved performance, systematic inflammation, and improved fecal microbiota composition of the broilers.