To analyze molecular characteristics and antimicrobial activity of bovine neutrophil β-defensin 12 (BNBD12), Pro-BNBD12 gene was amplified from Chinese Holstein cow using reverse transcription polymerase chain reaction. Based on the sequence of mature BNBD12 and codon preference in E. coli, a modified mature BNBD12 cDNA was synthesized, cloned into pET32a (+) vector and expressed in E. coli BL21 as a 26 kD fusion protein after isopropyl β-D-1-thiogalactopyranoside induction. The expressed mature BNBD12 accounted for 21.4% of total protein. 0.05 mg/ml purified BNBD12 had antimicrobial activity against both E. coli and S. aureus in vitro assayed by agar diffusion method. Electron microscopy found that BNBD12 treatment of E. coli and S. aureus could induce cell content leakage. Taking together, BNBD12 protein was successfully expressed in E. coli and it has antimicrobial activity against both Gram-positive and negative bacteria and potentials in control of mastitis clinically.