Background:　Hyperhomocysteinemia　(HHcy)　is　an　independent　risk　factor　for　cardiovascular　diseases　(CVDs).　Stachydrine　(STA)　is　an　active　component　in　Chinese　motherwort　Leonurus　heterophyllus　sweet,　which　has　been　widely　used　for　gynecological　and　cardiovascular　disorders.　This　study　is　aimed　to　examine　the　effects　of　STA　on　homocysteine　(Hcy)-induced　endothelial　dysfunction.　Methods:　The　effects　of　STA　on　vascular　relaxation　in　rat　thoracic　aortas　(TA),　mesenteric　arteries　(MA)　and　renal　arteries　(RA)　were　measured　by　using　Multi　Myograph　System.　The　levels　of　nitric　oxide　(NO),　tetrahydrobiopterin　(BH4)　and　guanosine　3＇,　5＇　cyclic　monophosphate　(cGMP)　were　determined.　Endothelial　nitric　oxide　synthase　(eNOS)　dimers　and　monomers　were　assayed　by　using　Western　blotting.　GTP　cyclohydrolase　1　(GTPCH1)　and　dihydrofolate　reductase　(DHFR)　expressions　were　measured　by　using　quantitative　reverse　transcriptase-PCR　(qRT-PCR)　and　Western　blotting.　Results:　STA　effectively　blocked　Hcy-induced　impairment　of　endothelium-dependent　vasorelaxation　in　rat　TA,　MA　and　RA.　STA-elicited　arterial　relaxations　were　reduced　by　NOS　inhibitor　NG-nitro-L-arginine　methyl　ester　(L-NAME)　or　the　NO-sensitive　guanylyl　cyclase　inhibitor　1H-　[1,　2,　4]　Oxadiazolo[4,3-a]quinoxalin-1-one　(ODQ),　but　not　by　inducible　iNOS　inhibitor　1400　W　nor　the　nonselective　COX　inhibitor　indomethacin.　Hcy　caused　eNOS　uncoupling　and　decreases　in　NO,　cGMP　and　BH4,　which　were　attenuated　by　STA.　Moreover,　STA　prevented　decreases　of　GTPCH1　and　DHFR　levels　in　Hcy-treated　BAECs.　Conclusion:　We　demonstrated　that　STA　effectively　reversed　the　Hcy-induced　endothelial　dysfunction　and　prevented　eNOS　uncoupling　by　increasing　the　expression　of　GTPCH1　and　DHFR.　These　results　revealed　a　novel　mechanism　by　which　STA　exerts　its　beneficial　vascular　effects.