Generally, Ca(OH)₂ pretreatment of lignocellulosics for fermentable sugar recovery requires a subsequent washing step for calcium removal and pH control for optimized saccharification. However, washing Ca(OH)₂-pretreated feedstock with water is considered problematic because of the low solubility of Ca(OH)₂ and its adsorption to biomass. In this study, we estimated the availability of carbonated water for calcium removal from the slurry of Ca(OH)₂-pretreated rice straw (RS). We tested two kinds of countercurrent washing sequences, four washings exclusively with water (W4) and two washings with water and subsequent two washings with carbonated water (W2C2). The ratios of calcium removal from pretreatment slurry after washing were 64.2 % for the W4 process and 92.1 % for the W2C2 process. In the W2C2 process, 49 % of the initially added calcium was recovered as CaO by calcination. In enzymatic saccharification tests under a CO₂ atmosphere at 1.5 atm, in terms of recovery of both glucose and xylose, pretreated, feedstock washed through the W2C2 process surpassed that washed through the W4 process, which could be attributed to the pH difference during saccharification: 5.6 in the W2C2 process versus 6.3 in the W4 process. Additionally, under an unpressurized CO₂ atmosphere at 1 atm, the feedstock washed through the W2C2 process released 78.5 % of total glucose residues and 90.0 % of total xylose residues. Thus, efficient removal of calcium from pretreatment slurry would lead to not only the recovery of added calcium but also the proposal of a new, simple saccharification system to be used under an unpressurized CO₂ atmosphere condition.