Alkaline stress is one of the abiotic stresses limiting cotton production. Though RNA-Seq analyses, have been conducted to investigate genome-wide gene expression in response to alkaline stress in plants, the response of sodium bicarbonate (NaHCO) stress-related genes in cotton has not been reported. To explore the mechanisms of cotton response to this alkaline stress, we used next-generation sequencing (NGS) technology to study transcriptional changes of cotton under NaHCO alkaline stress. A total of 18,230 and 11,177 differentially expressed genes (DEGs) were identified in cotton roots and leaves, respectively. Gene ontology (GO) analysis indicated the enrichment of DEGs involved in various stimuli or stress responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs associated with plant hormone signal transduction, amino acid biosynthesis, and biosynthesis of secondary metabolites were regulated in response to the NaHCO stress. We further analyzed genes enriched in secondary metabolic pathways and found that secondary metabolites were regulated to eliminate the reactive oxygen species (ROS) and improve the cotton tolerance to the NaHCO stress. In this study, we learned that the toxic effect of NaHCO was more profound than that of NaOH at the same pH. Thus, Na, HCO and pH had a great impact on the growth of cotton plant. The novel biological pathways and candidate genes for the cotton tolerance to NaHCO stress identified from the study would be useful in the genetic improvement of the alkaline tolerance in cotton.