Uremic pruritus is an unpleasant symptom in patients undergoing hemodialysis, and the underlying mechanisms remain unclear. β2-Microglobulin (β2-MG) is well-known as an MHC class I molecule and its level is increased in the plasma of patients undergoing hemodialysis. In this study, we investigated whether β2-MG was a pruritogen in mice. Intradermal injections of β2-MG into the rostral back induced scratching in a dose-dependent manner. Intradermal injection of β2-MG into the cheek also elicited scratching, but not wiping. β2-MG-induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone hydrochloride. β2-MG-induced scratching was not inhibited by antagonists of itch-related receptors (e.g., H1 histamine receptor (terfenadine), TP thromboxane receptor (DCHCH), BLT1 leukotriene B4 receptor (CMHVA), and proteinase-activated receptor 2 (FSLLRY-NH2)). However, β2-MG-induced scratching was attenuated in mice desensitized by repeated application of capsaicin and also by a selective transient receptor potential vanilloid 1 (TRPV1) antagonist (BCTC). In addition, β2-MG induced phosphorylation of extracellular signal-regulated kinase (a marker of activated neurons) in primary culture of dorsal root ganglion neurons that expressed TRPV1. These results suggest that β2-MG is a pruritogen and elicits itch-related responses, at least in part, through TRPV1-expressing primary sensory neurons.