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DNA binding with One Finger (DOF) transcription factor plays important roles in many aspects of plant growth and development, but the molecular mechanisms behinds its actions in plant development and stress tolerance are little known. Here, we report on the Brassica napus DOF gene, which is homologous to Arabidopsis Cycling DOF Factor1 and functions as a transcriptional regulator with regard to flowering time and freezing tolerance. The BnCDF1 gene was ubiquitously expressed in plants, and its expression was induced in response to low temperatures. Arabidopsis plants overexpressing BnCDF1 showed increased freezing tolerance. In addition, the expression of stress-responsive genes was activated in transgenic lines. Furthermore, overexpression in Arabidopsis of BnCDF1 delays flowering time by modulating the expression patterns of CO and FT flowering time control genes. In conclusion, the results reported herein connect BnCDF1 to the functions of freezing tolerance and flowering time.