Endothelial dysfunction, which leads to ischemic events under atherosclerotic conditions, can be attenuated by antagonizing the thromboxane-prostanoid receptor (TP) that mediates the vasoconstrictor effect of prostanoids including prostacyclin (PGI 2). This study aimed to determine whether antagonizing the E prostanoid receptor-3 (EP3; which can also be activated by PGI 2) adds to the above effect of TP deficiency (TP –/–) under atherosclerotic conditions and if so, the underlying mechanism(s). Atherosclerosis was induced in ApoE –/– mice and those with ApoE –/– and TP –/–. Here, we show that in phenylephrine pre-contracted abdominal aortic rings with atherosclerotic lesions of ApoE –/–/TP –/– mice, although an increase of force (which was larger than that of non-atherosclerotic controls) evoked by the endothelial muscarinic agonist acetylcholine to blunt the concurrently activated relaxation in ApoE –/– counterparts was largely removed, the relaxation evoked by the agonist was still smaller than that of non-atherosclerotic TP –/– mice. EP3 antagonism not only increased the above relaxation, but also reversed the contractile response evoked by acetylcholine in NO synthase-inhibited atherosclerotic ApoE –/–/TP –/– rings into a relaxation sensitive to I prostanoid receptor antagonism. In ApoE –/– atherosclerotic vessels the expression of endothelial NO synthase was decreased, yet the production of PGI 2 (which evokes contraction via both TP and EP3) evoked by acetylcholine was unaltered compared to non-atherosclerotic conditions. These results demonstrate that EP3 blockade adds to the effect of TP –/– in uncovering the dilator action of natively produced PGI 2 to alleviate endothelial dysfunction in atherosclerotic conditions.