Excessive use of neonicotinoid insecticides can cause various physiological and metabolic disorders in plants. Spermidine (Spd) plays a crucial role in mitigating plant abiotic stress. However, the mechanisms by which Spd regulates dinotefuran (DIN) tolerance in strawberries (Fragaria × ananassa Duch.) remain unclear. Here, we investigated the effects of foliar Spd on the DIN-induced stress in strawberries via a hydroponic experiment. Spd notably reduces the accumulation of DIN and its metabolites in roots, stems and leaves, particularly inhibiting DIN migration to the leaves. Spd also significantly improves plant growth of strawberries under DIN stress by enhancing photosynthetic capacity, increasing osmotic substance levels, reducing oxidative damage, and specifically regulating various enzyme activities and polyamine levels. Molecular docking reveals that non-covalent interactions dominate the binding processes of Spd and DIN with photosystem proteins D1, SOD, PAO, and ABC. Interestingly, transcriptomic analyses suggest that Spd competitively occupies the exclusive transport channels of DIN (e.g., ABC transporter subfamilies C and G) in DIN-induced strawberries. Additionally, Spd regulates the expression of genes related to hormone signaling pathways, leaf carbon metabolism, root phenylpropanoid metabolism, and activates the root MAPK pathway in strawberries, thereby enhancing plant stress resistance.