Mast cells play important roles in innate immunity through their activation via toll-like receptors (TLRs) but also contribute to neuroimmunological responses and inflammation through their activation by the neuropeptide substance P (SP) via Gα_i/o proteins. This study aims to compare the effects of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4) on SP-induced human mast cell activation. The human mast cell line LAD2 was employed and mast cell activation was determined by assays of β-hexosaminidase, IL-8 and intracellular calcium. TLR2 agonists did not cause degranulation, but induced the release of IL-8. Pretreatment of PGN and Pam3CSK4 inhibited SP induced degranulation but only Pam3CSK4 blocked SP induced calcium mobilization. SP-induced IL-8 release was synergistically enhanced by PGN but abolished by Pam3CSK4. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that synergistic release of IL-8 induced by PGN and SP involved calcineurin, ERK, NF-κB and PI3K signaling cascades whereas Pam3CSK4 inhibited SP induced mast cell activation by interfering with the interaction between SP and Gα_i/o proteins. These findings suggest that activation of human mast cells can be differentially modified by TLR2 agonists via distinct signaling pathways through facilitating formation of different TLR2 heterodimers with other TLRs.