Itch is an unpleasant sensation that induces the desire to scratch. Except for a sketchy map focusing on neural mechanisms underlying itch processing being drawn at the peripheral and spinal level over the past decade, the brain mechanisms remain poorly understood. Several previous studies indicated that anterior cingulate cortex (ACC) and prelimbic cortex (PrL), two subregions of the medial prefrontal cortex (mPFC) play an important role in regulating itch processing. However, the knowledge about whether infralimbic cortex (IL), another subregion of mPFC, is involved in modulating itch processing remains unclear. Here, we showed that the activity of IL excitatory pyramidal neurons was significantly elevated during itch-related scratching, and pharmacogenetic inhibition of IL pyramidal neurons significantly impaired itch-related scratching. Moreover, IL-medial striatum (MS) projections were verified as a critical neural pathway for modulating itch processing. Therefore, the present study firstly presents the regulatory function of IL pyramidal neurons during itch processing and also reveals that IL-MS projections are involved in modulating the itch processing.