Bacillus acidopullulyticus pullulanase (BaPul13A) is a widely used debranching enzyme in the starch industry. A few details have been reported on the heterologous expression of BaPul13A in Escherichia coli (E. coli). This study compares different E. coli expression systems to improve the soluble expression level of BaPul13A. When pET22b(+)/pET28a(+) was used as the expression vector, the soluble expression of BaPul13A can be achieved by tightly controlling basal expression, whereas pET-20b(+)/pGEX4T2 leads to insoluble inclusion bodies. An efficient process control strategy aimed at minimizing the formation of inclusion bodies and enhancing the production of pullulanase was developed by a step decrease of the temperature in a 5-L fermentor. The highest total enzyme activity of BaPul13A reached 1,156.32 U/mL. This work reveals that the T7 promoter with lac operator and lacI gene collectively contribute to the soluble expression of BaPul13A, whereas either a T7 promoter alone or combined with the lac operator and lacI gene results in poor solubility. Basal expression in the initial growth phase of the host significantly affects the solubility of BaPul13A in E. coli.