[Objective] We modified genes responsible for xylose metabolism and riboflavin biosynthesis in Bacillus subtilis. [Methods] Genes responsible for riboflavin biosynthesis were overexpressed or co-overexpressed with genes of xylose metabolism in Bacillus subtilis. Recombinant strains were evaluated by measuring the riboflavin yield and biomass. Fermentation was done in shake flask and fermenter to produce riboflavin with sucrose as sole carbon source or a mixture of sucrose/xylose. Meanwhile, riboflavin yield, xylose consumption and biomass were analyzed. [Results] Overexpression of ribA gene increased riboflavin yield by 99% and reduced maximum biomass by 30% because of autolysis. With ribA and ribH genes co-overexpression, riboflavin yield increased by 280% without biomass decrease and autolysis. Through a 70 h batch fermentation with 6.5% xylose and 1.5% sucrose as carbon source in a 5 L fermenter, riboflavin yield of 3.6 g/L was obtained, 80% higher than that with 8% sucrose as carbon source. Riboflavin yield decreased significantly by overexpressing the genes involved in xylose metabolism. [Conclusion] Co-overexpression of ribA and ribH genes avoided the cytotoxicity effectively and further increased riboflavin production.