Background The NADPH oxidase family (Nox) produces reactive oxygen species by adding the electron donated by NADPH to oxygen. Excessive reactive oxygen species production under a variety of pathological conditions has been attributed to increased Nox activity. Here, we aimed at investigating the role of Nox in cardiac ischemic injury through gain‐ and loss‐of‐function approaches. Methods and Results We modulated Nox activity in the heart by cardiac‐specific expression of Nox4 and dominant negative Nox4. Modulation of Nox activity drastically changes the cellular redox status. Increasing Nox activity by cardiac‐specific overexpression of Nox4 imposed oxidative stress on the myocardium [increased NAD (P) ⁺ / NAD (P)H and decreased glutathione/glutathione disulfide ratio] and worsened cardiac energetics and contractile function after ischemia‐reperfusion. Overexpression of the dominant negative Nox4 ( DN ), which abolished the Nox function, led to a markedly reduced state [decreased NAD (P) ⁺ / NAD (P)H and increased glutathione/glutathione disulfide ratio] at baseline and paradoxically promoted mitochondrial reactive oxygen species production during ischemia resulting in no recovery of heart function after reperfusion. Limiting the generation of reducing equivalent through modulating carbon substrates availability partially restored the NAD ⁺ / NADH ratio and protected dominant negative Nox4 hearts from ischemic injury. Conclusions This study reveals an important role of Nox in cardiac redox regulation and highlights the complexity of developing therapies that affect the intricately connected redox states.