Prostacyclin, also termed as prostaglandin I (PGI), evokes contraction in vessels with limited expression of the prostacyclin receptor. Although the thromboxane-prostanoid receptor (TP) is proposed to mediate such a response of PGI, other unknown receptor(s) might also be involved. TP knockout (TP) mice were thus designed and used to test the hypothesis. Vessels, which normally show contraction to PGI, were isolated for functional and biochemical analyses. Here, we showed that the contractile response evoked by PGI was indeed only partially abolished in the abdominal aorta of TP mice. Interestingly, further antagonizing the E-type prostaglandin receptor EP3 removed the remaining contractile activity, resulting in relaxation evoked by PGI in such vessels of TP mice. These results suggest that EP3 along with TP contributes to vasoconstrictor responses evoked by PGI, and hence imply a novel mechanism for endothelial cyclooxygenase metabolites (which consist mainly of PGI) in regulating vascular functions.