The underlying mechanisms imparting the growth phase-dependent acid tolerance have not been extensively investigated. In this study, we compared the acid resistance of the Bifidobacterium longum strain BBMN68 from different growth phases at lethal pH values ( pH 2.5, 3.0, and 3.5), and analyzed the activity of H+-ATPase, the composition of fatty acids, and the mRNA abundance of ffh, uvrA, recA, lexA, groES, and dnaK in cells from different growth phases. The results indicated that the survival rates of cells from early stationary ( ES) and late stationary ( LS) growth phases at lethal pH values were significantly higher than those of exponential growth phase cells. Our findings indicated that by inducing a continuously auto-acidizing environment during cell growth, the acid resistance of ES and LS cells was strengthened. The higher activity of H+-ATPase, the decrease in unsaturated fatty acids, and the increased expression of genes involved in DNA repair and protein protection in the cells in stationary growth phase were all implicated in the significantly increased acid resistance of ES and LS cells compared with exponential growth phase cells of the B. longum strain BBMN68.