Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. (No 2508). We previously showed that Xyl-B promoted endothelial NO release and protected against atherosclerosis through the Akt/eNOS pathway. Vascular NO production regulates vasoconstriction in central and peripheral arteries and plays an important role in blood pressure control. In this study, we examined whether Xyl-B exerted an antihypertensive effect in a hypertensive rat model, and further explored the possible mechanisms underlying its antihypertensive action. Administration of Xyl-B (20 mg·kg-1·d-1, ip, for 12 weeks) significantly decreased the systolic and diastolic blood pressure in a two-kidney, two-clip (2K2C) renovascular hypertensive rats. In endothelium-intact and endothelium-denuded thoracic aortic rings, pretreatment with Xyl-B (20 μmol/L) significantly suppressed phenylephrine (Phe)-induced contractions, suggesting that its vasorelaxant effect was attributed to both endothelial-dependent and endothelial-independent mechanisms. We used SNP, methylene blue (MB, guanylate cyclase inhibitor) and indomethacin (IMC, cyclooxygenase inhibitor) to examine which endothelial pathway was involved, and found that MB, but not IMC, reversed the inhibitory effects of Xyl-B on Phe-induced vasocontraction. Moreover, Xyl-B increased the endothelial NO bioactivity and smooth muscle cGMP level, revealing that the NO-sGC-cGMP pathway, rather than PGI2, mediated the anti-hypertensive effect of Xyl-B. We further showed that Xyl-B significantly attenuated KCl-induced Ca2+ entry in smooth muscle cells in vitro, which was supposed to be mediated by voltage-dependent Ca2+ channels (VDCCs), and reduced ryanodine-induced aortic contractions, which may be associated with store-operated Ca2+ entry (SOCE). Taken together, these findings demonstrate that Xyl-B exerts significant antihypertensive effects not only through the endothelial NO-sGC-cGMP pathway but also through smooth muscle calcium signaling, including VDCCs and SOCE.