Saline-alkali soil severely reduces the productivity of crops, including maize (Zea mays). Although several genes associated with saline-alkali tolerance have been identified in maize, the underlying regulatory mechanism remains elusive. Here, we report a direct link between colonization by arbuscular mycorrhizal fungi (AMF) and saline-alkali tolerance in maize. We identify s75, a natural maize mutant that cannot survive under moderate saline-alkali soil conditions or establish AM symbioses. The saline-alkali hypersensitive phenotype of s75 is caused by a 1340-bp deletion in Zm00001d033915, designated as ZmL75. This gene encodes a glycerol-3-phosphate acyltransferase localized in the endoplasmic reticulum, and is responsible for AMF colonization. ZmL75 expression levels in roots correspond with the root length colonization (RLC) rate during early vegetative development. Notably, the s75 mutant line shows a complete loss of AMF colonization, along with alterations in the diversity and structure of its root fungal microbiota. Conversely, overexpression of ZmL75 increases the RLC rate and enhances tolerance to saline-alkali soil conditions. These results suggest that ZmL75 is required for symbiosis with AMF, which directly improves saline-alkali tolerance. Our findings provide insights into maize-AMF interactions and offer a potential strategy for maize improvement.