Defensins are a class of small and diverse cysteine-rich proteins which have broad-spectrum antimicrobial activities. We identified and characterized a full-length cDNA encoding a putative defensin-like peptide from the whitefly Bemisia tabaci by RACE and quantitative real-time (qRT)-PCR. The full-length cDNA, named Btdef, was 388 bp long and contained an open reading frame of 228 bp. The putative mature Btdef had 46 amino acids with a molecular weight of 5.06 kDa. The deduced amino acid sequence showed significant homology with insect defensins from Heliothis virescens (76%) and Galleria mellonella (75%). The predicted mature form of Btdef was expressed as a recombinant peptide in Escherichia coli. Antimicrobial assays of the purified product indicated that Btdef was most active against fungi. qRT-PCR analyses indicated that Btdef mRNA was constitutively expressed in different tissues of B. tabaci, including fat body, midgut, ovaries and salivary gland, and was induced by fungal infection. Btdef mRNA expression was also significantly altered after feeding on different host plants, indicating that diet affects immune defences in B. tabaci. These results describe for the first time the basic properties of a defensin-like peptide from B. tabaci that probably plays an important role in the immune response against pathogens.