The CO2 balance of a commercial closed system with artificial lighting (CSAL), in which lettuce plants (Lactuca sativa L. ‘Early Impulse’, ‘King Crown’, and ‘Cos Lettuce’) were produced every day and CO2 was added to the air by gas cylinders and workers’ respiration, was analyzed. In the experiment, 95% of the CO2 supplied from cylinders was apparently assimilated by the lettuce plants in the commercial CSAL, suggesting that the supplied CO2 was used efficiently. The amounts of CO2 assimilated by the lettuce plants and loss resulting from leakage, respectively, accounted for 78% and 22% of the total amount of CO2 supplied. The amounts of CO2 supplied by the cylinders and by the workers’ respiration, respectively, accounted for 83% and 17% of the total amount of CO2 supplied. Based on the analysis, a relatively high CO2 utilization efficiency of 78% was observed in the experiment despite the operation rate of 33%, which is defined as the percentage of the culture beds with plants. If the operation rate could be increased to 100%, the CO2 utilization efficiency would reach 92%. These results showed that CO2 supplied by the workers’ respiration helped to reduce the amount of CO2 supplied by the cylinders and hence the CO2 cost in a commercial CSAL.