Ticks, as blood sucking arthropods, are able to transmit various pathogens. Their immune system involves many antimicrobial molecules to fight against them. Among these antimicrobials, defensins, a 5.3 kDa peptides, play an important role in rapid immune answer. In this study we examined the antimicrobial spectrum of Dermacentor marginatus defensin (def DM) with respect to the fact that Dermacentor variabilis ticks are not able to successfully maintain and transmit Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. Expression of the def DM gene was detected in hemolymph, midgut and salivary glands. Defensin was isolated from hemolymph using RP-HPLC and its sequence was determined by mass spectrometry and Edman degradation. Synthetic mature peptide def DM revealed an anti-Gram-positive bacterial role as well as borreliacidal activity, with concentration dependent influence. These results suggest a possible role in the clearing of borrelia spirochetes ingested by D. marginatus ticks.