Orexin-A and Orexin-B play important roles in many physiological processes in which Orexins orchestrate diverse downstream effects via two G-protein coupled receptors: Orexin1R and Orexin2R. Two alternative C-terminus splice variants of the mouse Orexin receptors mOX2alphaR and mOX2betaR have recently been identified. This study explored the possibility of heterodimerization between mOX2alphaR and mOX2betaR, and investigated novel signal transduction characteristics after stimulation. The dimerization of mOX2alphaR and mOX2betaR was confirmed by BRET and co-immunoprecipitation assays. Meanwhile, in HEK293 cells, co-expression of mOX2alphaR and mOX2betaR resulted in a strengthened increase in activation of ERK1/2, with maximal activation at 5 min and 100 nM. Furthermore, heterodimerization also elicits stronger intracellular Ca2+ elevation after Orexin(s) stimulation, followed by a slower decline in intracellular Ca2+ to a steady endpoint Protein Kinase C Inhibitor significantly inhibited these downstream effects. In addition, the cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase and the T-lymphocyte activation of nuclear factor-responsive element reporter activity were significantly up-regulated after Orexin(s) stimulation. Besides, Orexin-A/-B induced a significantly higher rate of HEK293 cell proliferation in cells co-expressing mOX2alphaR/mOX2betaR compared to the control group. Taken together, we provide conclusive evidence that mOX2alphaR can form a functional heterodimer with mOX2betaR and this leads to increased PKC and decreased protein kinase A activity by ERK signal pathway leading to a significant increase in cell proliferation. The nature of this signaling pathway has significant implications for the role of Orexin in the regulation of physiological processes including the homeostasis of feeding.