Background: Nebivolol is a β1-adrenergic receptor (β1-AR) antagonist, inducing endothelial nitric oxide (NO) release, most likely due to β3-AR agonism. Nebivolol is vasculoprotective and cardioprotective in the setting of hypertension. In this study, we investigated the effects of nebivolol, compared with those of bisoprolol, on vascular and myocardial function in diabetic mice. Methods: Diabetic (Leprdb/db) and nondiabetic mice (Leprdb/+) were treated with vehicle, nebivolol, or bisoprolol for 16 weeks. Endothelium-dependent and endothelial-independent relaxations were studied in isolated aortic segments. Myocardial twitch performance was studied in isolated right ventricular papillary muscles. Results: In aortic segments of diabetic mice, endothelium-dependent relaxations were significantly shifted to the right. This shift was not prevented by chronic nebivolol or bisoprolol treatment. Papillary muscle twitches of diabetic mice displayed a significant delay in the onset of relaxation, and an increased time from peak active force to 50% relaxation, leading to prolonged twitch activity, without changes in twitch amplitude. These changes were not prevented in nebivolol- and bisoprolol-treated diabetic mice. Surprisingly, in nebivolol-treated mice, regardless of diabetic status, twitch duration was further increased. Applying nebivolol to papillary muscles in the organ bath reproduced the changes induced by chronic treatment in vivo; this was not the case for bisoprolol. Nebivolol-induced changes were blunted by a β3-AR antagonist and by NO synthase (NOS) inhibition. Conclusions: Diabetes-induced changes in vascular and myocardial function were not prevented by treatment with nebivolol or bisoprolol. Strikingly, myocardial effects of nebivolol differed from those of bisoprolol, by inducing a β3-AR and NOS-dependent prolongation of twitch activity.