Abstract: To investigate the mechanism underlying early flowering in tobacco caused by chilling stress, NtDUF599, a candidate gene related to the phenomenon was screened by RNA-seq analysis. NtDUF599 encoded a protein containing 236 amino acids with unknown functions, and its expression was highly induced by chilling stress. A RNAi plasmid, pBWA (V) HS-NtDUF599-RNAi vector, was constructed and a total of 10 different transgenic lines were generated by Agrobacterium-mediated transformation. Seedlings of non-transgenic and transgenic plants were treated with chilling stress (12 ℃) for 10 days and kept grown until bud stage under normal growth condition. We found that the emergence of flower buds of NtDUF599-RNAi transgenic plants was approximately 10 days earlier than non-transgenic plants. Furthermore, leaf numbers of NtDUF599-RNAi transgenic plants were less than those of non-transgenic plants. Those results revealed for the first time that DUFs (Domain of unknown functions) were involved in the chilling-induced early flowering in tobacco.