Aim 12/15‐lipoxygenase (12/15‐LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15‐LO metabolites 12(S)‐hydroxyeicosatetraenoic acid (HETE), 15‐HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15‐trihydroxyeicosatrienoic acids (11,12,15‐THETAs) as endothelium‐derived relaxing factors. However, the observed 12‐LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12‐LO activity. Methods Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A3, trioxilin A3 and trioxilin C3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα ‐HEK). Results All identified vascular 12‐LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A3, C3 and hepoxilin A3 (3 μm) relaxed arteries constricted with the thromboxane mimetic, U46619‐constricted arteries (maximum relaxations of 78.9 ± 3.2, 29.7 ± 4.6, 82.2 ± 5.0 and 88.0 ± 2.4% respectively), but not phenylephrine‐constricted arteries. In TPα‐HEK cells, trioxilin A3, C3 and hepoxilin A3 (10 μm) inhibited U46619 (10 nM)‐induced increases in intracellular calcium by 53.0 ± 7.2%, 32.8 ± 5.0% and 37.9 ± 13.5% respectively. In contrast, trioxilin B3 and hepoxilin B3 were not synthesized in arteries and exhibited little biological activity. Conclusion Trioxilin A3 and C3 and hepoxilin A3 are endogenous vascular relaxing factors. They are not endothelium‐derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist‐induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists.