Cadmium (Cd) contamination in maize poses a significant threat to global food security due to its persistent accumulation in crops. In this study, the effects of foliar application of glycerol on Cd accumulation in maize seedlings were studied. Our results demonstrated that under Cd treatment, biomass, total chlorophyll content, net photosynthetic rate (Pn), Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity, Phosphoenolpyruvate carboxylase (PEPC) activity, sucrose levels, and carbohydrate levels in maize seedlings significantly increased after glycerol application. H₂O₂ and MDA levels in both the aboveground and belowground portions of the maize plants significantly decreased. Moreover, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in the aboveground parts significantly increased. Notably, maize plants used glycerol to chelate Cd, which was fixed within the cell wall and soluble fraction of the roots, reducing Cd transport to the shoots and significantly lowering the Cd transport coefficient (TF). Transcriptomic data suggested that glycerol-mediated alleviation of Cd stress in maize seedlings may be associated with phenylpropanoid biosynthesis, plant-pathogen interactions and photosynthesis pathways. These molecular patterns align with the observed physiological improvements. This study provided a novel approach to effectively alleviate excessive Cd in maize and suggested possible applications of glycerol in cultivating plant resistance to heavy metals.