NP-1815-PX is a selective P2X4 receptor antagonist. Administration of a P2X4 receptor antagonist to asthma model mice improves the asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effect of these antagonists on tracheal smooth muscles (TSMs) has not been investigated. This study examined the effect of NP-1815-PX on guinea pig TSM contraction. In epithelium-intact TSMs, NP-1815-PX strongly suppressed ATP-induced contraction, which was suppressed by indomethacin or ONO-8130 (an EP₁ receptor antagonist). NP-1815-PX strongly suppressed U46619 (a TP receptor agonist)- and PGF_2α-induced epithelium-removed TSM contractions, which were largely inhibited by SQ 29,548 (a TP receptor antagonist). Additionally, NP-1815-PX strongly suppressed the U46619-induced increase in intracellular Ca²⁺ concentration in TP receptor-expressing cells. In contrast, NP-1815-PX did not substantially inhibit TSM contractions induced by carbachol, histamine, neurokinin A, and 50 mM KCl. These findings indicate that: 1) NP-1815-PX inhibits guinea pig TSM contractions mediated through the TP receptor in addition to P2X4 receptor whose stimulation induces EP₁ receptor-related mechanisms, and 2) NP-1815-PX may be a useful therapeutic for asthma.