•The differential expression genes of industrial hemp were identified and analyzed under NaHCO3 stress.•The major pathways response to NaHCO3 stress were identified.•The hub genes response to NaHCO3 stress were identified. This paper reports the gene expression profiling of industrial hemp exposed to NaHCO3 stress. In this study, RNA-sequencing (RNA-seq) was used to investigate gene expression profiling of industrial hemp roots exposed to 100 mM NaHCO3 (for a period of 0, 1, 6, and 12 h). The results revealed that some pathways (plant hormone signal transduction and synthesis, phenylpropanoid biosynthesis, and metabolic pathways of starch, sucrose, nitrogen, and amino acids) may be related to the response of industrial hemp under NaHCO3 stress. Furthermore, the sixteen modules were identified by weighted gene co-expression network analysis (WGCNA), of which six were significantly associated with NaHCO3 stress. These six modules were basically enriched in pathways associated with endocytosis, starch and sucrose metabolism, nitrogen metabolism, and phenylpropanoid biosynthesis. And the hub genes of key pathways were associated with GTP-binding protein, glutamate synthase, trehalose-phosphate, glycosyltransferase, and lignin synthesis. The results elucidate the molecular mechanism of industrial hemp involved in response to NaHCO3 stress.