Increasing evidence shows that sex hormones exert a protective effect on the vasculature, especially in the regulation of the active vasomotor responses. However, whether sex hormones affect vascular remodeling is currently unclear. In the present study, we tested the hypothesis that testosterone in males and β-estradiol in females prevent inward remodeling, possibly through inhibition of cross-linking activity induced by enzymes of the transglutaminase family (TG). Small mesenteric arteries were isolated from male and female Wistar rats. Dose dependent relaxation to testosterone and β-estradiol was inhibited by the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), confirming that these hormones induce nitric oxide release. When arteries were cannulated, pressurized, and kept in organ culture with endothelin-1 for three days we observed strong vasoconstriction and inward remodeling. Remodeling was significantly inhibited by testosterone in males, and by β-estradiol in females. This preventive effect of sex hormones was not observed in the presence of L-NAME. Inward remodeling was also reduced by the inhibitor of TG L682.777, both in males and females. In arteries from female rats, endothelin-1 increased TG activity, and this effect was prevented by β-estradiol. L-NAME induced a significant increase in TG activity in the presence of sex hormones in arteries from both genders. We conclude that testosterone and β-estradiol prevent constriction-induced inward remodeling. Inward remodeling, both in males and females, depends on NO and TG activity. In females, inhibition of inward remodeling could be mediated by NO-mediated inhibition of TG activity.