Despite the increasing prevalence of hypoxia in natural habitats occupied by the American paddlefish, basal bony fish, and ram ventilator, information about its response to hypoxia is scarce. To understand the physiological and biochemical responses of juvenile paddlefish (~150 g) to acute (<24 h) and chronic hypoxia (≥24 h), blood oxygen transport, blood acid-base balance, and metabolic stress were evaluated under four different partial pressures of oxygen [pO; normoxia (148 mmHg), mild hypoxia (89 mmHg), moderate hypoxia (59 mmHg), and extreme hypoxia (36 mmHg)], all at 21 °C. Arterial blood samples were collected from paddlefish after they had been exposed to treatments for 0.25, 2, 6, 24, and 72 h, and analyzed for hematocrit, pO, total oxygen content, oxygen saturation, pCO, pH, hemoglobin, Na, K, Ca, Cl, glucose, and lactate. Mild hypoxia only caused a reduction in blood pO and oxygen saturation. Both acute and chronic moderate and extreme hypoxia caused a decrease in blood pH, pO, total oxygen content, plasma Na, and Cl at all time points. Acute moderate and extreme hypoxia resulted in an increase in blood pCO, plasma glucose, lactate, and hematocrit. Chronic exposure to moderate hypoxia resulted in an increase in plasma lactate, red blood cell count, and hemoglobin. This study shows that paddlefish are able to physiologically compensate for mild hypoxia, but exhibit secondary stress responses and are unable to return to homeostasis when exposed to both acute and chronic moderate hypoxia, and die after 3-8 h of extreme hypoxia.