G protein coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interaction in the clinically important thromboxane A2 receptor (TP) and prostacyclin receptor (IP) is poorly understood. TP activates its cognate G-protein (Gαq) in response to binding of thromboxane, while IP signals through Gαs in response to the binding of prostacyclin. Here we utilized a combination of approaches consisting of, chimeric receptors, molecular modeling and site-directed mutagenesis to precisely study G protein coupling specificity. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both sequence and length of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors, TP and IP, that can switch G protein specificities. The validity of the chimeric technique and the derived molecular model was confirmed by introducing clinically relevant naturally occurring mutations (TP R60L and IP R212C). Our findings provide new molecular insights into the prostanoid receptor-G protein interactions, of general significance, for understanding the structural basis of G protein activation by GPCRs in health and cardiovascular disease.