The objective of this study was to determine if mechanisms involved in vascular dysfunction in type 2 diabetes differ with sex. Vascular reactivity, expression, and activation of rhoA and rho kinase were measured in aorta from male and female nondiabetic C57BLKS/J and diabetic BKS.Cg-m(+/+) Lepr(db)/J (db/db) mice, a model of type 2 diabetes. Relaxation to acetylcholine and nitroprusside was similar in aorta from nondiabetic male and female mice. Relaxation to acetylcholine was reduced approximately 50% in both male and female diabetic mice. Although inhibition of rho kinase with H-1152 increased relaxation to acetylcholine and nitroprusside in nondiabetic males, it had no effect on the response in either nondiabetic or diabetic females or diabetic males. Contraction to serotonin was increased similarly in male and female diabetic mice compared with nondiabetic mice and was reduced following inhibition of rho kinase with either fasudil or H-1152. Activation of rhoA and its downstream effector, rho kinase, was greater in aorta from diabetic males compared with nondiabetic males. In contrast, there were no differences in vascular activation of rhoA or rho kinase in diabetic females. The increased activity of rhoA and rho kinase in diabetic mice was not due to a change in protein expression of rhoA or rho kinase (ROCK1 and ROCK2) in vessels from either males or females. Although contractile dysfunction in vessels occurs in both male and female diabetic mice, the dysfunction in diabetic males is dependent upon activation of rhoA and rho kinase. Alternative mechanisms affecting rho kinase activation may be involved in females.