Introduction: Erythropoietin (EPO)-induced hypertension is a common adverse event among patients receiving EPO therapy and is associated with risk of myocardial infarction and stroke, yet the underlying molecular mechanisms responsible for EPO related cardiovascular disease mortality are not clear. Regulator of G-protein signaling-5 ( Rgs5 ) is highly expressed in vascular smooth muscle cells (SMCs) and its action in fine tuning G-protein signaling is critical in regulating vascular tone. Whether Rgs5 plays a role in EPO-mediated hemodynamic changes is not known. We have previously shown that increased arterial pressure after EPO administration in mice is associated with significant reduction in aortic Rgs5 expression. One of the proposed mechanisms of Rgs5 mediated cellular response involves regulation of MAPK/ERK signaling pathway. Here, we investigated the role of Rgs5 and ERK1/2 pathway signaling in EPO-induced hypertension in mice. Methods: 10-12 weeks old male and female C57BL/6 mice were randomly divided into two groups: (1) Vehicle (0.9% saline-VEH), (2) EPO, (N=8). VEH and EPO were administered intraperitoneally (EPO 75U/30g, 3 times/week) for 20 days. Rgs5 silencing was performed in mouse aortic SMCs (N=3), and phophoryated-ERK1/2 (p-ERK1/2) was quantified by western blot. Results: EPO treated mice showed increased p-ERK1/2 activity in aorta and heart (fold change in aorta = 1.37, P = 0.001; fold change in heart = 1.72, P = 0.015) suggesting MAPK activation as possible mediator of EPO-induced increase in blood-pressure. In vitro , EPO increased the p-ERK activity in mouse aortic SMCs (MOVAS VEH 0.99 ± 0.05, EPO 1.31 ± 0.13, P =0.032) and interestingly Rgs5 silencing in MOVAS exacerbated EPO-induced p-ERK activation ( Rgs5 siRNA + EPO 1.70 ± 0.11, P =0.038 vs EPO), suggesting a potential role of Rgs5 in EPO mediated ERK activation and thus in regulating vascular tone. Conclusions: Our data indicated that Rgs5 is crucial in regulating EPO-induced ERK1/2 activation and that Rgs5 and its signaling pathway may serve as novel treatment targets in reducing cardiovascular complications in patients receiving EPO therapy.