With the development of industrialization process in society, the problem of cadmium (Cd) pollution in soil is increasing. However, Nicotiana tabacum has a strong Cd enrichment capacity in leaves, which seriously affects its economic value. To investigate the mechanism by which tobacco responds to Cd stress, tobacco leaves were harvested from the culture solution with Cd concentrations of 0 μmol L-1 and 500 μmol L-1 for subsequent transcriptome sequencing. In this study, a total of 76.94 Gb clean data was obtained, with Q30 base percentage exceeding 95.43%. The results showed that 7735 differentially expressed genes (DEGs) were screened under Cd stress conditions, including 4833 up-regulated genes and 2902 down-regulated genes. The reliability of transcriptome data was verified by qRT-PCR analysis to detect the expression patterns of candidate gene. Gene ontology (GO) annotation as well as Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed on differentially expressed transcripts. GO functional enrichment revealed that the differentially expressed genes were mainly distributed in metabolic processes, response to stimulus, cellular anatomical entity, catalytic activity, and transcription regulator activity. Meanwhile, KEGG analysis showed that the up-regulated differentially expressed genes were mainly involved in biosynthesis of amino acids, carbon metabolism, oxidative phosphorylation, and citrate cycle. Down-regulated differentially expressed genes were primarily enriched in photosynthesis, biosynthesis of secondary metabolites, metabolic pathways, and plant hormone signal transduction. Further analysis of plant hormone signal transduction pathways revealed that there were eight plant hormone pathways involved in response to cadmium stress in tobacco, and the relative expression patterns of different hormone gene member were also different. Experimental results from plant hormone application on tobacco leaves demonstrated that the regulation of gibberellins, brassinosteroids, and jasmonic acid pathways played roles in tobacco's response to cadmium stress. The experimental results of Arabidopsis hormone signal mutant showed that plants respond to cadmium stress by regulating ethylene, gibberellin, brassinosteroid, and jasmonic acid pathways. In conclusion, this study not only explores the regulatory network of tobacco resistance to Cd stress, but also lays a theoretical foundation for the genetic improvement of crop resistance.