Accurate and timely glucose monitoring is essential in intensive care units. Real-time continuous glucose monitoring system (CGMS) has been advocated for many years to improve glycemic management in critically ill patients. In order to determine the effect of calibration time on the accuracy of CGMS, real-time subcutaneous CGMS was used in 18 critically ill patients. CGMS sensor was calibrated with blood glucose measurements by blood gas/glucose analyzer every 12 hours. Venous blood was sampled every 2 to 4 hours, and glucose concentration was measured by standard central laboratory device (CLD) and by blood gas/glucose analyzer. With CLD measurement as reference, relative absolute difference (mean +/- SD) in CGMS and blood gas/glucose analyzer were 14.4% +/- 12.2% and 6.5% +/- 6.2%, respectively. The percentage of matched points in Clarke error grid zone A was 74.8% in CGMS, and 98.4% in blood gas/glucose analyzer. The relative absolute difference of CGMS obtained within 6 hours after sensor calibration (8.8% +/- 67.2%) was significantly less than that between 6 to 12 hours after calibration (20.1% +/- 13.5%, p < 0.0001). The percentage of matched points in Clarke error grid zone A was also significantly higher in data sets within 6 hours after calibration (92.4% versus 57.1%, p < 0.0001). In conclusion, real-time subcutaneous CGMS is accurate in glucose monitoring in critically ill patients. CGMS sensor should be calibrated less than 6 hours, no matter what time interval recommended by manufacturer.