B7-H3 an immune-checkpoint molecule, a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. An anti-B7-H3 antibody omburtamab has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Elicitation of cellular mechanism behind the anti-B7-H3 blockade is important to reveal the immune-independent functions of B7-H3. Warburg effect is one feature of metabolic dysregulation during malignant transformation. To investigate the effect of anti-B7-H3 blockade in the regulation of glucose metabolism in cancer cells, we measured dynamic ECAR and OCR as indicatives of glycolysis and oxidative respiration in NSCLC cells. We demonstrated that anti-B7-H3 antibody blockade switched cell metabolism from glycolysis to oxidative phosphorylation. The alteration of glucose metabolism probably was associated with cellular ROS-mediated pathway. These results clearly showed that, in addition to immune-regulatory function, B7-H3 possessed immune-independent functions in NSCLC. The study is supported by the Science and Technology Development Fund, Macau SAR (FDCT/0015/2918/A1).