Nicotinamide adenine dinucleotide (NAD⁺) not only transfers electrons in mitochondrial respiration, but also acts as an indispensable cosubstrate for Sirt1, the class III histone/nonhistone deacetylase. However, NAD⁺ is depleted in myocardial ischemia/reperfusion (IR) injury. The objective of this study was to investigate the role of exogenous NAD⁺ supplementation in hypoxia/reoxygenation (HR)‐stressed H9c2 cardiac myoblasts. Firstly, the effects of distinct treating time points and doses of NAD⁺ supplementation on the viability of HR‐stressed H9c2 cells were detected. Secondly, intracellular NAD⁺ levels in HR‐stressed H9c2 cells at various extracellular NAD⁺ concentrations were determined. Thirdly, the role of NAD⁺ supplementation in HR‐induced cell apoptosis and its relevance to Sirtuin 1‐p53 pathway were investigated. Exogenous NAD⁺ supplementation elevated intracellular NAD⁺ level and reduced HR‐induced cell death in both time‐ and concentration‐dependent manners. It appeared that NAD⁺ supplementation exerted the greatest protection when extracellular concentration ranged from 500 to 1000 μm and when NAD⁺ was added immediately after reoxygenation began. NAD⁺ replenishment restored Sirt1 activity, reduced the acetylation level of p53 (Lys373 & 382), and attenuated cell apoptosis in HR‐stressed H9c2 cells, whereas inhibition of Sirt1 activity alleviated the effects of NAD⁺ replenishment. These results indicated that exogenous NAD⁺ supplementation attenuated HR‐induced cell apoptosis, which was at least partly mediated by restoring Sirt1 activity and subsequently inhibiting p53 activity via deacetylating p53 at lysine 373 and 382.