Abstract: Topping is one of the important technical procedures of Nicotiana tabacum production. In order to study the gene expression patterns at the topping stage in Nicotiana tabacum, we selected the Daning tobacco growing area in Guangxi and collected ten tissue samples at the topping stage. The samples were used to extract total RNAs and to construct stranded specific total RNA sequencing library and to sequence using the Illumina HiSeq 2000 sequencer. Through quality control, more than 91 million clean reads were obtained and a total of 50706 genes were detected to be expressed from ten tissues at the topping stage. After normalization, screening for tissue-specific and genes differentially expressed among tested tissues, a total of 687 genes were identified to be tissue-specifical and 444 genes showed differential expression before and after topping. Above data and results will not only provide some support for exploring the molecular responses of topping, but also provide molecular targets for the regulatiion of Nicotiana tabacum vegetative and reproductive growth.