Pinolenic acid (PA), an 18 : 3 n-6 polyunsaturated fatty acid abundant in pine nut oil, is a structural isomer of γ-linolenic acid (GLA) and α-linolenic acid (ALA). Notably, GLA and ALA have been reported to inhibit prostanoid TP receptor-mediated vascular contraction. Whether PA similarly modulates TP receptor-dependent responses, however, remains unknown. In the present study, we investigated the effects of PA on TP receptor-mediated contraction in porcine coronary arteries (PCAs), TP receptor-evoked intracellular Ca2+ responses in human TP receptor-expressing cells, and its potential binding mode using molecular docking. In endothelium-denuded PCAs, PA (3-30 μM) concentration-dependently inhibited contractions induced by the TP receptor agonist U46619 and prostaglandin F2α (PGF2α), whereas it had little effect on contractions evoked by depolarization (high-KCl) or by other vasoactive agonists. PA caused a parallel rightward shift of the U46619 concentration-response curve, and Schild analysis yielded a slope not significantly different from unity; the corresponding pA2 value was 4.91. In human TP receptor-expressing 293T cells, PA significantly attenuated U46619-induced intracellular Ca2+ elevation, whereas it did not affect PGF2α-induced Ca2+ responses in prostanoid FP receptor-expressing cells. Docking simulations based on the human TP receptor crystal structure (Protein Data Bank ID: 8XJO) suggested that PA may occupy the orthosteric binding pocket, with its carboxylate group positioned near His89. Collectively, these findings suggest that PA inhibits TP receptor-mediated contraction and exhibits pharmacological properties consistent with competitive antagonism, supporting the concept that C18:3 fatty acids may modulate TP receptor signaling.