A 1050 bp up-stream regulatory fragment of the transcription factor gene NAC1 in Arabidopsis thaliana was isolated using polymerase chain reaction (PCR) based techniques. The fragment was used to substitute the 35S promoter of the pBI121 plasmid to construct a β-glucuronidase gene (GUS) expression system. The construct was introduced into tobacco (Nicotiana tabaccum) plants by the Agrobacterium-mediated transferring method. GUS expression pattern was studied by using the transgenic lines. The results showed that the GUS driven by the NAC1up-stream regulatory region was specifically expressed in the root meristem region, basal areas of the lateral root primordium and the lateral roots. The GUS expression was induced by 3-indolebutyric acid (IBA) and gibberellins (GA3 and GA4+7). The results indicated that the up-stream regulatory fragment of NAC1 responded to plant hormones. The fragment might be involved in both auxins and gibberellins signaling in promoting the development of lateral roots.