Background: Central venous oxygen saturation (ScvO2) is an essential test readily performed both by medical and nursing personnel in a critical care setting. It gives information on the patient‘s oxygen supply, oxygen consumption, and cardiac output. It plays an important role in early goal-directed treatment. Objectives: This study was planned to assess the effect of different fractions of inspired oxygen (FiO2) levels on central venous oxygen saturation for consideration during the evaluation of central venous oxygen saturation. Methods: This interventional cross-over study enrolled 60 critically ill, nonmechanically ventilated patients. Blood samples were repeatedly drawn from the distal end of the central venous catheter for blood gas analysis after administration of 30%, 40%, and 50% FIO2 respectively. Results: The results showed that increasing FiO2 from 30% to 40% resulted in a mean increase in ScvO2 of 6.2%. While increasing FiO2 from 40% to 50% resulted in a mean increase in ScvO2 of 3.2%. A significant increase in ScvO2 with changes in FiO2 level was recorded among patients in shock or patients with pneumonia (from 30% to 50%, p=0.002 in shock patients and from 30% to 40%, p=0.02 in patients with pneumonia). Conclusion: Increasing FiO2 resulted in a substantial rise in ScvO2. ScvO2 changes in response to a therapeutic challenge should be interpreted at constant FiO2 level, especially in patients with pneumonia.