Escherichia coli expressing the levansucrase SacB gene cannot grow on LB plates containing 5% sucrose, therefore this gene has been used as a counter-selection marker in many studies. However, the mechanism of this phenomenon is still unclear. In this study, we compared the characteristics of E. coli strains expressing SacB with and without signal peptide. We observe that only SacB with signal peptide causes sucrose intolerance of E. coli. In addition, kinetic studies and product analyses indicated that the signal peptide exerts no obvious effect on the enzymatic properties of SacB. Therefore, the signal peptide-dependent protein translocation pathway is clearly crucial for the sucrose intolerance of SacB-expressing E. coli. Additionally, E. coli containing SacB minus signal peptide forms swollen colonies on sucrose-containing LB plates which may provide a convenient and high-throughput screening method for directed evolution of levansucrases.