Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel β-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (ORF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of β-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the β-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka β-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fold up-regulation of the β-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka β-defensin. A proximal 100 base pair (bp) sequence (+26 to −73) and the next 1700 bp sequence (−73 to −1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor κ B (NF-κB) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5′ flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka β-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka β-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-κB and Sp1.