Abstract: Dehydration responsive element binding protein (DREB) is one of the important transcription factors in plants, playing a key role in plant growth and development as well as in response to abiotic stress. In this study, based on a previous genome-wide identification of the SiDREB gene family in foxtail millet, a candidate gene SiDREB39 that was significantly induced by low temperature was screened. To explore the role of the foxtail millet SiDREB39 gene in plant low-temperature response, the SiDREB39 gene was cloned from Jingu 21. Bioinformatic analysis, subcellular localization, expression pattern analysis, and functional validation were performed by constructing an overexpression vector and transforming it into Arabidopsis thaliana, followed by phenotypic observation, physiological index measurement, and transcriptome sequencing. The results showed that the full-length CDS of SiDREB39 was 660 bp, encoding 219 amino acids. RT-qPCR analysis revealed that SiDREB39 exhibited the highest expression level in the leaves of Jingu 21. The SiDREB39 protein was localized in the nucleus and cytoplasm. Under low-temperature stress, overexpression of SiDREB39 significantly increased the seed germination rate and plant survival rate of transgenic Arabidopsis, while reducing ion leakage rate and malondialdehyde (MDA) content. Antioxidant enzyme activities were markedly enhanced, soluble protein and proline contents increased, reactive oxygen species (ROS) accumulation decreased, and the expression levels of cold-responsive genes were upregulated. RNA-seq analysis identified 990 differentially expressed genes (DEGs) between low-temperature-treated transgenic Arabidopsis and wild-type Arabidopsis (LT-OE2 vs LT-WT). Among these, several key genes involved in ROS signaling and scavenging (e.g., AT2G28190, AT5G05410) were significantly induced in the transgenic plants. In conclusion, overexpression of SiDREB39 enhances plant tolerance to low-temperature stress by enhancing antioxidant enzyme activities and osmotic regulation ability, as well as inducing the expression of cold-responsive genes. This study provides an important candidate gene and theoretical basis for cold-resistant molecular breeding in foxtail millet.