The soluble transhydrogenase (UdhA) is one of two transhydrogenases that play a role in maintaining the balance between NAD(H) pools and NADP(H) pools in Escherichia coli Although UdhA has been extensively used in metabolic engineering and biocatalysis for cofactor regeneration, its role in acid resistance has not been reported. Here we used DNA microarray to explore the impact of UdhA on transcript levels. We demonstrated that during growth on acetate, the expression of genes involved in the respiratory chain and Gad acid resistance system was inhibited in the udhA-knockout strain. The deletion of udhA significantly repressed the expression of six genes (gadA, gadB, gadC, gadE, hdeA and hdeB) which are involved in Gad acid resistance and resulted in low survival of the bacterium at a low pH of 4.9. Moreover, UdhA was essential for NADH production which is important for the adaptive growth of E. coli on acetate, while NADH concentration in the udhA-knockout strain was quite low and supplemental NADH significantly increased the expression of acid resistance genes and survival of the udhA-knockout strain. These results demonstrated that UdhA is an important source of NADH of E. coli growth on acetate and affects Gad acid resistance system under acetate stress.