Zinc/iron-regulated transporter proteins (ZIPs) are involved in the transport of zinc and iron, maintenance of ion homeostasis, and regulation of plant responses to abiotic stresses. Although previous studies have identified members of the ZIP gene family and screened for alkaline-responsive ZIP genes in wild soybean (Glycine soja), the functional implications of these genes under alkaline stress conditions remain to be fully elucidated. The present study aimed to identify the GsZIP7 gene and investigate its regulatory role in Arabidopsis and wild soybean under alkaline stress. The results showed that GsZIP7 was highly expressed in roots and young stems. GsZIP7 exhibited differential expression in response to alkaline stress, zinc deficiency, or iron deficiency in wild soybean. In addition, heterologous expression of GsZIP7 in yeast mutants increased tolerance to iron- and zinc-deficient conditions. Overexpression of GsZIP7 in Arabidopsis resulted in reduced root growth, decreased fresh weight, and a weakened antioxidant defense system. Furthermore, GsZIP7-overexpressing soybean hairy roots showed increased sensitivity to alkaline stress, whereas GsZIP7-RNAi lines exhibited enhanced tolerance. Together, these findings indicate that GsZIP7 negatively regulates alkaline tolerance in plants.