After an ischemia and reperfusion (I/R) injury, reestablishing blood flow to the infarct region can salvage myocardium by clearing apoptotic cells, removing scar tissue, stimulating angiogenesis, and recruiting progenitor cells for tissue regeneration. Stimulating the growth of new microcirculation in the infarct area can help revive cardiac function. Angiogenesis is a process of developing new blood vessels from the existing vessels. Our previous work on Dual specificity phosphatase 4 (DUSP4) demonstrated that DUSP4-/- hearts sustain a larger infarct and have poor functional recovery, when isolated hearts were subjected to I/R. Uncontrolled p38 activation is the main effectors for this functional alteration.In this study, DUSP4 overexpression in endothelial cells was used to investigate the role of DUSP4 in the development of vascular function, and its role against oxidant stress. DUSP4 overexpression promotes angiogenesis and tube formation (increased by 57.3% ± 6.2% versus control; p < 0.0001). Immunoblotting reveals that DUSP4 overexpression down-regulates p53 (0.59 ± 0.04 versus 1.0 ± 0.01 of control) expression, which modulates HIF1a pathway and angiogenesis. Moreover, NO is a critical and small molecule involved in regulating vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. The level of NO generation determined using DAF fluorescence microscopy demonstrated that DUSP4 overexpression augments NO generation. The increase in NO generation also correlates to the increase in eNOS protein and mRNA (5.84 ± 0.37 versus control) expression. DUSP4 overexpression decreases H/R-induced cleaved caspase-3 activation to the similar level of the control. Together, these results demonstrated that DUSP4 is a critical protein involving vascular function and promoting angiogenesis via p53 and eNOS pathways. This will improve the future application of tissue regeneration by targeting the DUSP4 gene and serving a valuable therapeutic option for the treatment of ischemic heart disease via promoting angiogenesis.