Wild-type B. subtilis strain W168 was de novo engineered for inosine biosynthesis. Inactivation of deoD and purA led to 0.15 ± 0.04 and 6.44 ± 0.39 g inosine/l yields, respectively. The deoD purA double mutant accumulated 7.6 ± 0.34 g inosine/l, with a 4.7% (w/w) conversion ratio from glucose to inosine. Comparative metabolic flux analysis revealed that the fluxes from inosine to hypoxanthine and from inosine monophosphate to adenosine monophosphate in the double mutant decreased to 14.0 and 0.61% of those in the wild-type strain. The major role of purA was demonstrated when inactivation of deoD and purA were found to contribute additively to inosine accumulation. This work is expected to contribute to the improvement of the fermentative production of purine nucleosides in the microbial industry.