Pruritus is a common symptom of dermatological disorders and has a major negative impact on QOL. Previously, it was suggested that human β-defensin peptides elicit itch through the activation of mast cells. In this study, we investigated in more detail the mechanisms by which β-defensins induce itch by defining the receptors activated by these peptides in humans and mice, by establishing their action in vivo, and by examining their expression in inflammatory dermal diseases. We found that elevated expression of DEFB103 is highly correlated with skin lesions in psoriasis and atopic dermatitis. We showed that the peptide encoded by this gene and related genes activate Mas-related G protein-coupled receptors with different potencies that are related to their charge density. Furthermore, we establish that although these peptides can activate mast cells, they also activate sensory neurons, with the former cells being dispensable for itch reactions in mice. Together, our studies highlight that specific β-defensins are likely endogenous pruritogens that can directly stimulate sensory neurons.