β-Adrenoceptors (β-ARs) play a critical role in the regulation of cardiovascular function. Intracellular oxygen homeostasis is crucial for the survival of cardiomyocytes. However, it is still unclear whether β-AR activation can modulate intracellular oxygen. Here we used mitochondrial and cytosolic target Renilla luciferase to detect intracellular oxygen concentration. Pharmacological experiments revealed that β2-AR activation specifically regulates intracellular oxygen in cardiomyocytes and COS7 cells. This effect was abrogated by inhibitory G protein (Gi) inhibition, endothelial nitric oxide synthase (eNOS) blockade, and NO scavenging, implicating that the β2-AR-Gi-eNOS pathway is involved in this regulation. β2-AR activation increased the AMP/ATP ratio, AMPK activity, ROS production and prolyl hydroxylase activity. These effects also contribute to the regulation of β2-AR signalling, thus providing an additional layer of complexity to enforce the specificity of β1-AR and β2-AR signalling. Collectively, the study provides novel insight into the modulation of oxygen homeostasis, broadens the scope of β2-AR function, and may have crucial implications for β2-AR signalling regulation.