Adenylosuccinate synthetase (AdSS), encoded by purA, is generally considered as one of the rate-limiting enzymes involved in the de novo biosynthesis of adenosine. In this study, effect of purA overexpression on adenosine production was investigated and Bacillus subtilis XGL-XY and XGL-A were constructed. B. subtilis XGL-XY harboring a recombinant plasmid pBEA containing purA showed 25.5 % and 29.2% increased adenosine production and yield but a decline in cell growth. Considering the metabolic burden caused by pBEA, an additional purA gene under the control of P43 promoter was integrated into the B. subtilis XGL genome in purA locus by single crossover, resulting in B. subtilis XGL-A. The strain exhibited improved adenosine production, yield and similar cell growth with B. subtilis XGL. Furthermore, lowered IMP concentration was detected in B. subtilis XGL-XY and B. subtilis XGL-A. Fed-batch fermentation assay showed that, compared with B. subtilis XGL, 29.4 % and 18.3 % increase in adenosine production and yield and up to 17.3 g/L and 0.207 g/g glucose was achieved by B. subtilis XGL-A. Above all, it can be concluded that AdSS is a crucial enzyme in adenosine synthesis and purA overexpression could drove more metabolic flux from IMP to adenosine synthesis and thus enhance adenosine production.