AbstractAtopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. In this study, topically applied substance P (SP) significantly alleviated AD‐like clinical symptoms in 2, 4, 6‐trinitrochlorobenzene (TNCB)‐induced dermatitis in NC/Nga mice. This effect was nullified by pretreatment of the neurokinin‐1 receptor (NK‐1R) antagonist CP99994. SP treatment significantly reduced the infiltration of mast cells and CD3‐positive T cells as well as inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and thymic stromal lymphopoietin (TSLP), in AD‐like skin lesions and decreased the levels of IgE and thymus and activation‐regulated chemokine in serum. This SP‐induced alleviation of allergic inflammatory responses was also confirmed as reduced activation in the axillary lymph nodes (aLN) and spleen, suggesting the systemic effect of SP on immune responses in TNCB‐induced NC/Nga mice. Furthermore, SP‐mediated TSLP reduction was confirmed in human keratinocyte culture under pro‐inflammatory TNF‐α stimulation. Taken together, these results suggest that topically administered SP may have potential as a medication for atopic dermatitis.