An antimicrobial peptide gene named OnBD, which belongs to the β-defensin family, was cloned in Nile tilapia (Oreochromis niloticus) using the expressed sequence tag (EST) cloning method. The putative 66-residue OnBD prepeptide consists of two domains: an N-terminal 24-residue signal domain (signal peptide) and a 42-residue C-terminal antimicrobial domain (mature peptide). Annotation of the antimicrobial domain architecture showed that the OnBD peptide contains the signature motif and six conserved cysteines, a characteristic similar to that of β-defensins in other fish. The OnBD mature peptide shared other common features of antimicrobial peptides, such as being amphipathic and cationic (+3 charge) and having a small molecule (less than 100 a.a.). We also used real-time RT-PCR to investigate the expression pattern of the OnBD gene. The results showed that mRNA of OnBD could be detected in various tissues including the blood, gills, head kidney, intestine, liver, muscle, skin and spleen of Nile tilapia. Quantitative analysis revealed that skin had the highest expression level. Minimum inhibitory concentrations (MICs) of the chemically synthesized OnBD were examined against eight different aquatic bacterial pathogens and shown to exhibit potent antimicrobial activities to all of them, especially to Edwardsiella tarda and Streptococcus iniae, the two most important pathogens to tilapia, with the MIC reaching as low as 4 µM. The molecular characteristics and bioactivities of OnBD suggested that this antimicrobial peptide may play an important role in the innate immunity of Nile tilapia and that it has potential applications in the treatment of bacterial infections.