Cold temperatures often severely restrict the growth, distribution and productivity of plants. The freezing tolerance of plants from temperate climates can be improved by undergoing periods of cold acclimation (CA). Tobacco is an important economic plant and is sensitive to cold stress. However, the dynamic changes and regulatory mechanisms of gene expression and metabolic processes during CA remain largely unknown. In this study, we performed RNA sequencing and metabolomic profiling analyses to identify the genes and metabolites specifically expressed during CA. Our transcriptomic data revealed 6905 differentially expressed genes (DEGs) during CA. Functional annotation and enrichment analyses revealed that the DEGs were involved mainly in signal transduction, carbohydrate metabolism and phenylpropanoid biosynthesis. Moreover, a total of 35 significantly changed metabolites were identified during CA via an LC-MS platform. Many protective metabolites, such as amino acids, carbohydrates, tricarboxylic acid (TCA) cycle intermediates and phenylpropanoid-related substances, were identified during CA. The gene-metabolite network extensively outlined the biological processes associated with the utilization of sugars, activation of amino acid metabolism, TCA cycle and phenylpropanoid biosynthesis in tobacco under CA. The results of our present study provide a comprehensive view of signal transduction and regulation, gene expression and dynamic changes in metabolites during CA.