Pullulanase is crucial to the specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in the starch-processing industry. Recombinant Bacillus subtilis that employs an inducible promoter would be a suitable candidate for pullulanase expression because of its safety and controllable production, but its level of pullulanase activity is relatively low. In this study, we investigated the effect of the enhancers DegQ, DegU, and DegS on pullulanase expression in a recombinant B. subtilis inducible system. The genes degQ, degU, and degS were introduced to the recombinant plasmid pMA0911-P-sacB-pul harboring the promoter P-sacB. signal peptide LipA, and gene encoding pullulanase. The regulatory effects of the enhancers involved in recombinant plasmids on pullulanase expression level were evaluated in B. subtilis WB600 and WB800, respectively. The positive regulation of DegQ toward pullulanase expression was detected from B. subtilis WB800, leading to a 60% increase in enzyme activity. In addition, enzyme activity was further enhanced by inserting the degQ gene to the position closer to the promoter P-sacB. Consequently, pullulanase activity reached 26.5 U ml(-1) from the B. subtilis WB800/pMA0911-P-sacB- pul-degQ(N) after expression optimization, which was a 5.9-fold increase compared to that of the original strain B. subtilis WB800/pMA0911-P-saCB-pul. Hence, the inducible expression of the enzyme was efficiently enhanced by regulating the enhancer DegQ from recombinant B. subtilis WB800.