Itch is a common symptom of chronic skin diseases including atopic dermatitis (AD). The mechanism of itch in AD remains poorly understood, and there are few effective treatments to prevent AD itch. Mas-related G protein-coupled receptor A3 (MrgprA3) has attracted attention as a marker of primary sensory neurons that specifically transmit itch signals, but it is still unclear whether MrgprA3-expressing neurons are involved in itch in AD. We have developed an AD itch mouse model by repeatedly applying house dust mite (HDM) extract to the skin with barrier dysfunction. We generated a transgenic mouse strain, in which the human diphtheria toxin receptor (DTR) gene was situated under the control of the Mrgpra3 promoter, to determine the roles of the MrgprA3-expressing neurons in itch behavior. When a diphtheria toxin was administered, a majority of DTR⁺/MrgprA3⁺ neurons were depleted in the dorsal root ganglion. Acute itch responses after HDM application were suppressed under this condition, while spontaneous and touch-evoked itch behaviors remained intact. Our results suggest that the MrgprA3-expressing primary sensory neurons should mediate acute itch response induced by allergen challenge in AD mice.