Abstract: GsARHP, which encodes hypothetical protein up-regulated by alkaline stress, was identified from RNA-seq data of wild soybean (Glycine soja) at the early stage of the salt stress response. Real-time PCR analyses showed that GsARHP was induced by alkali stress. A bioinformatics analysis showed that the gene encodes a hydrophilic protein consisting of 130 amino acids, with a signal peptide but no transmembrane domain. A plant overexpression vector was constructed and transformed into alfalfa by Agrobacterium tumefaciens-mediated cotyledonary node infection. Three transgenic lines were identified by PCR, Southern blotting, and RT-PCR analyses. The non-transgenic lines became wilted and yellow or even died under 0, 100, and 150 mmol/L NaHCO₃ treatment for 14 days, while the transgenic lines grew well under the same conditions. Further analyses showed that the transgenic lines had significantly lower relative plasma membrane permeability and malondialdehyde (MDA) content than did non-transgenic lines (P<0.01), and significantly higher chlorophyll content and CAT activity (P<0.01). These results indicated that overexpression of GsARHP could enhance the alkali resistance of alfalfa.