Activation of PKCβ2 induced by hyperglycemia contributes to impaired angiogenesis in endothelial cells. The purpose of the present study was to investigate whether PKCβ2 inhibition can attenuate the hyperglycemia-induced impaired angiogenesis of myocardium to improve cardiac function following myocardial infarction (MI) in diabetes. In vitro, human umbilical vein endothelial cells (HUVECs) were cultured in low glucose (L-G) (5.6 mmol/l) or high glucose (H-G) (33.3 mmol/l) medium in the presence or absence of LY333531 (LY333) (10 nmol/l), a selective PKCβ2 inhibitor. In vivo, with the use of an MI diabetic rat model, animals were randomized to receive LY333 (10 mg/kg/day) orally for 4 weeks after MI, or no treatment whatsoever. Treatment of HUVECs with LY333 prevented the H-G-induced decrease of tube formation, migration and proliferation. Furthermore, exposure of HUVECs to H-G activated PKCβ2 and decreased levels of phospho-Akt (p-Akt) and phospho-endothelial nitric oxide synthase (p-eNOS) expression, which was also prevented by LY333. Compared with MI rats without therapy, LY333-treated MI rats showed an increase in left ventricular ejection fraction (LVEF) and fractional shortening (FS), but a reduction in infarct size. Furthermore, treatment of rats with LY333 not only significantly increased the capillary density of ischemic myocardium, but also significantly elevated the levels of p-Akt and p-eNOS expression. We also observed a significant increase of VEGF expression in myocardium measured by immunostaining in MI and LY333 groups compared to sham group. Anti-CD31 immunostaining revealed that MI rats treated with LY333 exhibited increased density of capillaries compared with sham group rats. However, treatment of rats with LY333 did not result in significant increases in vascular endothelial growth factor (VEGF) expression at both the mRNA and protein levels in myocardium, and in the plasma level of VEGF compared with MI rats without therapy. Overall, these results suggest that inhibition of PKCβ2 may be a novel therapeutic approach for preserving cardiac function after MI, in part by improving impaired angiogenesis of myocardium in diabetes.