During the fermentation of D-ribose with glucose and calcium gluconate mixture by Bacillus subtilis mutant D-756, the cell growth was improved and the yield of D-ribose was significantly increased through the feeding of gluconate as pH regulator. And the effect on the key enzymes involved in D-ribose production was studied. The activity of gluconate kinase in unit fermentation volume could be enhanced by addition of gluconate. In addition, the cells growth and D-ribose accumulation were not affected under weak alkaline condition. Consequently the fermentation pH was maintained at 7.2 by gluconate and the yield of D-ribose reached 76.8 g/L after incubation for 72 h.