To develop cost-effective systems for D-lactate production, here, the effect of high-cell density cultivation of metabolically engineered Lactobacillus plantarum on D-lactate production was evaluated. A xylose-assimilating strain of L. plantarum was anaerobically cultured with mixed sugars (glucose and xylose) as substrates. Compared to undiluted nutrient-rich de Man, Rogosa, and Sharpe (MRS) medium, D-lactate production by cultivating in 10-fold diluted MRS (0.1 MRS) medium or normal saline solution was 89.7 and 81.3 %, respectively. Notably, the xylose consumption rate was comparable in the three cultures, whereas the glucose consumption rate decreased by 18.3 and 26.1 % in 0.1 MRS medium and normal saline solution, respectively, resulting in a reduction of the D-lactate production rate. The D-lactate productivity in high-cell density cultivation was proportional to the initial cell concentrations. The use of a two-step cultivation process involving growing and resting cells in a single bioreactor revealed that the ratio of the glucose and xylose consumption rates (based on grams consumed) in resting cell conditions was 1.88, whereas that in growing conditions was 2.58. Cultivation of L. plantarum in growing conditions for 24 h produced 73.2 g/l D-lactate with the yield of 0.90 g/g, whereas cells cultivation under resting cell conditions in a saline solution for 24 h produced 68.7 g/l D-lactate with the yield of 0.93 g/g. In total, 141.9 g/l D-lactate was produced after 48 h cultivation, a value that represents the highest reported concentration of D-lactate produced from mixed sugars to date. Our findings contribute to the cost-effective, large-scale production of D-lactate.