In this study, the drought-responsive gene HvFBX158 from barley was transferred to Arabidopsis thaliana, and overexpression lines were obtained. The phenotypic characteristics of the transgenic plants, along with physiological indicators and transcription level changes of stress-related genes, were determined under drought treatment. Under drought stress, transgenic plants overexpressing HvFBX158 exhibited enhanced drought tolerance and longer root lengths compared to wild-type plants. Additionally, malondialdehyde and hydrogen peroxide contents were significantly lower in transgenic lines, while superoxide dismutase activity was elevated. Quantitative RT-PCR showed that the expression levels of drought and stress response genes, including AtP5CS, AtDREB2A, AtGSH1, AtHSP17.8, and AtSOD, were significantly upregulated. Transcriptome analysis further confirmed that HvFBX158 regulated multiple stress tolerance pathways. In summary, the overexpression of the HvFBX158 gene enhanced drought tolerance in Arabidopsis thaliana by regulating multiple stress response pathways. This study provides a practical basis for improving drought-resistant barley varieties and lays a foundation for subsequent research on F-box family genes for stress resistance in barley.