Background and Purpose: Despite availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in pre-clinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 induces intracellular Ca2+, mitogen activated protein kinase (MAPK) and -arrestin signaling the airway, leading to both inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 reduction with C391 would attenuate allergen-induced asthma indicators in murine models. Experimental Approach: We evaluated the ability for C391 to alter Alternaria alternata-induced PAR2 signaling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced asthma indicators in vivo in two murine strains. Key Results: C391 blocked A. alternata-induced, PAR2-dependent Ca2+ and MAPK signaling in 16HBE14o- cells, as well as -arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute asthma murine models. Conclusions and Implications: To our knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs.