Aim: RhoA/Rho-associated kinase and arginase are implicated in vascular complications in diabetes. This study investigated whether RhoA/Rho-associated kinase and arginase inhibition protect from myocardial ischaemia-reperfusion injury in type 1 diabetes and the mechanisms behind these effects. Methods: Rats with streptozotocin-induced type 1 diabetes and non-diabetic rats were subjected to 30min myocardial ischaemia and 2h reperfusion after being randomized to treatment with (1) saline, (2) RhoA/Rho-associated kinase inhibitor hydroxyfasudil, (3) nitric oxide synthase inhibitor N-G-monomethyl-l-arginine monoacetate followed by hydroxyfasudil, (4) arginase inhibitor N-omega-hydroxy-nor-l-arginine, (5) N-G-monomethyl-l-arginine monoacetate followed by N-omega-hydroxy-nor-l-arginine or (6) N-G-monomethyl-l-arginine monoacetate given intravenous before ischaemia. Results: Myocardial arginase activity, arginase 2 expression and RhoA/Rho-associated kinase activity were increased in type 1 diabetes (p<0.05). RhoA/Rho-associated kinase inhibition and arginase inhibition significantly reduced infarct size in diabetic and non-diabetic rats (p<0.001). The cardioprotective effects of hydroxyfasudil and N-omega-hydroxy-nor-l-arginine in diabetes were abolished by nitric oxide synthase inhibition. RhoA/Rho-associated kinase inhibition attenuated myocardial arginase activity in diabetic rats via a nitric oxide synthase-dependent mechanism. Conclusion: Inhibition of either RhoA/Rho-associated kinase or arginase protects from ischaemia-reperfusion injury in rats with type 1 diabetes via a nitric oxide synthase-dependent pathway. These results suggest that inhibition of RhoA/Rho-associated kinase and arginase constitutes a potential therapeutic strategy to protect the diabetic heart against ischaemia-reperfusion injury.