[Background] The exchange rate of substances inside and outside cells has a significant effect on the transformation activity of whole-cells. [Objective] To increase the yield of γ-aminobutyric acid (GABA) synthesized by whole-cell transformation, we investigated the improving effect of ethanol on the apparent activity of whole-cell glutamate decarboxylase (GAD) in Enterococcus faecium. [Methods] We explored the mechanism of ethanol in improving the apparent activity of whole-cell GAD by comparing the effects of ethanol on the enzymatic properties of pure GAD and whole-cell GAD, cell structure, and membrane permeability. [Results] Low-concentration ethanol significantly promoted the activity of whole-cell GAD with the optimal concentration of 7.5%. At this concentration, the apparent activity of whole-cell GAD was increased by 41.63%, and the optimal reaction pH of whole-cell GAD did not change. However, the sensitivity of whole-cell GAD to the changes of extracellular pH and the Michaelis constant (K_m) of whole-cell GAD were both reduced. This concentration of ethanol had no significant effect on pure GAD (P>0.05), and did not cause GAD leakage or cell damage. In addition, 7.5% ethanol increased the activity of GAD only when it coexisted with the cells in reaction system. The results indicated that 7.5% ethanol altered the cell permeability and enhanced the mass transfer rate of cells, thereby improving the apparent activity of whole-cell GAD. [Conclusion] Due to the low price and high safety, ethanol demonstrates great potential of application in the improvement of whole-cell GAD activity for the industrial production of GABA.