Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid abundant in fish oil. Chronic administration of DHA has been reported to improve asthma due to its anti-inflammatory effects. However, the immediate effects of DHA on isolated tracheal smooth muscle (TSM) contractility have not been studied. In this study, we investigated the potential inhibitory effects of DHA on the guinea pig TSM contractions. DHA (3 × 10⁻⁵ M) significantly inhibited TSM contractions induced by U46619 (a thromboxane A₂ (TXA₂) mimetic, 10⁻⁸ M) and prostaglandin F_2α (PGF_2α, 5 × 10⁻⁷ M). The TSM contractions induced by U46619 (10⁻⁸ M) and PGF_2α (5 × 10⁻⁷ M) were significantly inhibited by SQ 29,548 (a prostanoid TP receptor antagonist, 10⁻⁶ M). DHA (4 × 10⁻⁵ M/6 × 10⁻⁵ M) shifted the concentration-response curves (CRCs) for U46619 and PGF_2α to the right in a concentration-dependent manner. However, the slope of the regression line in the Schild plot of DHA vs. U46619/PGF_2α was larger than unity. In contrast, DHA (4 × 10⁻⁵ M) did not significantly affect the CRCs for acetylcholine, histamine, and leukotriene D₄. These findings indicate that DHA selectively inhibit prostanoid TP receptor-mediated TSM contractions. DHA may have preventive and ameliorative effects on asthma attacks associated with TSM hyper-contraction caused by TXA₂ and PGF_2α.