ABSTRACTDrug resistance against bacteria and fungi has become common in recent years, and it is urgent to discover novel antimicrobial peptides to manage this problem. Many antimicrobial peptides from insects have been reported to have antifungal activity and are candidate molecules in the treatment of human diseases. In the present study, we characterized an antifungal peptide named blapstin that was isolated from the Chinese medicinal beetleBlaps rhynchopeteraused in folk medicine. The complete coding sequence was cloned from the cDNA library prepared from the midgut ofB. rhynchopetera. It is a 41-amino-acid diapause-specific peptide (DSP)-like peptide stabilized by three disulfide bridges and shows antifungal activity againstCandida albicansandTrichophyton rubrumwith MICs of 7 μM and 5.3 μM, respectively. TheC. albicansandT. rubrumtreated with blapstin showed irregular and shrunken cell membranes. In addition, blapstin inhibited the activity ofC. albicansbiofilm and showed little hemolytic or toxic activity on human cells and it is highly expressed in the fat body, followed by the hemolymph, midgut, muscle, and defensive glands. These results indicate that blapstin may help insects fight against fungi and showed a potential application in the development of antifungal reagents.IMPORTANCECandida albicansis one of the conditional pathogenic fungi causing severe nosocomial infections.Trichophyton rubrumand other skin fungi are the main pathogens of superficial cutaneous fungal diseases, especially in children and the elderly. At present, antibiotics such as amphotericin B, ketoconazole, and fluconazole are the main drugs for the clinical treatment ofC. albicansandT. rubruminfections. However, these drugs have certain acute toxicity. Long-term use can increase kidney damage and other side effects. Therefore, obtaining broad-spectrum antifungal drugs with high efficiency and low toxicity for the treatment ofC. albicansandT. rubruminfections is a top priority. Blapstin is an antifungal peptide which shows activity againstC. albicansandT. rubrum. The discovery of blapstin provides a novel clue for our understanding of the innate immunity ofBlaps rhynchopeteraand provides a template for designing antifungal drugs.