Aims: To explore the molecular mechanism of the carbon catabolite derepression in Lactobacillus fermentum 1001 when this strain consumed xylose and glucose simultaneously. Methods and Results: The transcriptional level of ccpAf was measured by real-ime qPCR, revealing that ccpAf transcribed mRNA normally in Lact. fermentum 1001. The ccpAf gene could complement the ccpA-deficiency of a Lactococcus lactis mutant. Moreover, when the phosphofructokinase from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was expressed in Lact. fermentum 1001, the recombinant preferred glucose to fructose rather than to xylose. All data suggested that CcpAf was functional in Lact. fermentum 1001. In addition, the promoter (Plx) activity of the xyl operon from Lact. fermentum 1001 was further test in Lactobacillus casei BL23, and it could drive the expression of green fluorescent protein in the presence of glucose. Conclusions: The ability of Lact. fermentum 1001 to co-utilize xylose and glucose resulted from the deficiency of catabolite responsive element in P1x rather than the null mutation of the ccpAf gene. Significance and Impact of the Study: Lactobacillus fermentum 1001 is a potential candidate as a CCR-absent cell factory to transform biomass to high-value-added products. P1x was provided for engineering LAB to enhance fermentation efficiency by avoiding CCR.