Background: Laparoscopic surgery has several benefits, but it requires prolonged carbon dioxide (CO2) insufflation. Several factors affect the accuracy of continuous and noninvasive hemoglobin (SpHb) monitoring, but the effects of CO2 insufflation are undetermined. This study investigated the effect of CO2 insufflation on SpHb monitoring in laparoscopic surgery. Material/Methods: Twenty patients undergoing laparoscopic gastrectomy were enrolled. Anesthesia was maintained using sevoflurane and remifentanil within an end-tidal CO2 of 30-45 mmHg. The CO2 insufflation was maintained at 12 mmHg using CO2. SpHb was monitored with a Radical-7 Pulse CO-Oximeter, and laboratory hemoglobin (tHb) was analyzed using a satellite blood analyzer. Results: Forty paired measurements were analyzed. The mean perfusion index, SpHb, and tHb were 3.10±1.77%, 10.92±1.48 g/dL, and 11.51±0.88 g/dL, respectively. SpHb underestimated tHb with a bias (precision) of -0.59 (1.28 g/dL), and the 95% limit of agreement was wide (-3.11 to 1.92 g/dL). SpHb was moderately correlated with tHb (r=0.50, 95% CI: 0.23 to 0.70). The concordance rate was 67%. DSpHb was not correlated with DtHb (r=0.29, 95% CI: -0.18 to -0.65). A similar bias, wider limits of agreement, a higher |SpHb-tHb|, but more significant correlation between SpHb and tHb were observed for the “PaCO2 <40 mmHg” range compared with the “40 mmHg £PaCO2” range. Conclusions: SpHb may have an acceptable accuracy but has a weak trending ability in the presence of CO2 insufflation, and it can be affected by PaCO2. Further research on the effects of CO2 insufflation on SpHb is needed.