DREB1/C-repeat binding factor (CBF) is a plant-specific family of transcription factors and plays a crucial role in freeze tolerance. In the present work, two groups of drought-responsive element binding factor (DREB)-like genes were isolated from Brassica napus, named Group I and Group II. The two groups of genes were both induced by low temperature, but the expression of Group I preceded that of Group II. The Group I DREBs could specifically bind with the DRE cis-acting element and activate the expression of downstream genes, but Group II factors were trans-inactive although they still had the ability to bind with DRE, which was confirmed by electrophoretic mobility shift assay. Fluorescence quenching assays indicated that the DRE binding ability of the two groups was similar. Co-expression of Group II could depress the trans-activation activity of Group I DREB in a concentration-dependent manner. These results strongly suggested that the trans-active Group I DREBs were expressed at the early stage of cold stress to open the DRE-mediated signaling pathway in cold stress, whereas the trans-inactive Group II DREBs were expressed at the later stage to close the signal pathway in a competitive manner. The results herein provide a new insight into the regulation mechanisms of the DRE-mediated signaling pathway in response to cold stress.