Antimicrobial peptides produced by Lactococcus lactis are natural and safe. The aim of this study was to investigate the physicochemical properties of antimicrobial peptides produced by L. lactis L-H410 and optimize the fermentation conditions for its production. The antimicrobial peptides exhibited broad-spectrum activity, and was stable against a broad pH range (2.0–9.5) and thermal treatment (at 80 ℃ for 30 min) and susceptible to proteases. The fermentation conditions (temperature, initial pH, and inoculum size) and medium components (carbon sources, nitrogen sources, stimulating factors, and divalent salt ions) were optimized by the combined use of one-factor-at-a-time method, Plackett-Burman design, path of steepest ascent, Box-Behnken design, and response surface methodology. The optimum fermentation conditions that provided maximum relative antibacterial potency of 378.7 AU/mL, which was 1.2 times of that before optimization (318.4 AU/mL), were determined as 3.32%, 2.5%, 0.2%, 0.02%, 29 ℃, 6.95, and 2.5% for sucrose, peptone, polyethylene glycol (PEG) 8000, manganese sulphate concentrations (m/m), temperature, initial pH, and initial inoculum, respectively.