D-Ribose is a five-carbon sugar used for the commercial synthesis of riboflavin, antiviral agents, and flavor enhancers. Batch fermentations with transketolase-deficient B. subtilis JY1 were carried out to optimize the production of D-ribose from xylose. The best results for the fermentation were obtained with a temperature of $37^{circ}C$ and an initial pH of 7.0. Among various sugars and sugar alcohols tested, glucose and sucrose were found to be the most effective for both cell growth and D-ribose production. The addition of 15 g/l xylose and 15 g/l glucose improved the fermentation performance, presumably due to the adequate supply of ATP in the xylose metabolism from D-xylulose to D-xylulose-5-phosphate. A batch culture in a 3.7-1 jar fermentor with 14.9 g/l xylose and 13.1 g/l glucose resulted in 10.1 g/l D-ribose concentration with a yield of 0.62 g D-ribose/g sugar consumed, and 0.25 g/l-h of productivity. Furthermore, the sugar utilization profile, indicating the simultaneous consumption of xylose and glucose, and respiratory parameters for the glucose and sucrose media suggested that the transketolase-deficient B. subtilis JY1 lost the glucose-specific enzyme II of the phosphoenolpyruvate transferase system.