Although psychological stress has been anecdotally related to flare-ups of vitiligo, the underlying mechanism(s) has remained poorly defined. In this study, we investigated whether substance P (SP) and its proteolytic fragments (SP1-9 and SP1-7) modulate the anti-melanocyte immune response under stress. Dual immunofluorescence staining revealed increased SP+PGP9.5+ sensory innervation and avidin⁺ mast cell infiltration in active vitiligo lesions and in the ear skin of mice subjected to chronic restraint stress (CRS). Using a vitiligo model where mice were immunized with the Tyrp-2180-188 peptide, we demonstrated that MrgprB2-dependent mast cell activation is essential for SP- or CRS-driven disease exacerbation. In vivo intradermal administration revealed that full-length SP and the SP1-9 fragment activate mast cells and worsen depigmentation, but the SP1-7 fragment did not. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed that SP proteolytic cleavage by keratinocyte-derived MMP9, rather than by fibroblast-derived neprilysin, generates elevated levels of SP1-9, which in turn activates mast cells via MRGPRX2 (mouse ortholog MrgprB2). These findings establish the SP-MRGPRX2 signaling axis as a critical link between psychological stress and vitiligo progression.