LL-37 is a cationic peptide belonging to the cathelicidin family that has antimicrobial and immune-modulatory properties. Here we show that the mammalian mono-ADP-ribosyltransferase-1 (ART1), which selectively transfers the ADP-ribose moiety from NAD to arginine residues, ADP-ribosylates LL-37 in vitro. The incorporation of ADP-ribose was first observed by Western blot analysis and then confirmed by MALDI-TOF. Mass-spectrometry showed that up to four of the five arginine residues present in LL-37 could be ADP-ribosylated on the same peptide when incubated at a high NAD concentration in the presence of ART1. The attachment of negatively charged ADP-ribose moieties considerably alters the positive charge of the arginine residues thus reducing the cationicity of LL-37. The cationic nature of LL-37 is key for its ability to interact with cell membranes or negatively charged biomolecules, such as DNA, RNA, F-actin and glycosaminoglycans. Thus, the ADP-ribosylation of LL-37 is expected to have the potential to modulate LL-37 biological activities in several physiological and pathological settings.