Differential distribution of neonicotinoids (NEOs) in albumen, yolk, and eggshell is a critical factor influencing their bio-accumulative behavior and the subsequent human health risks. However, there is currently no relevant research available. We collected 62 egg samples from 31 sampling sites across China and analyzed the concentrations and characteristics of 12 parents NEOs (p-NEOs) and 8 metabolites NEOs (m-NEOs) in albumen, yolk, and eggshell. NEOs were frequently detected in differentiated egg matrices, with the highest concentrations observed in Northeast China. The concentrations of m-NEOs were generally higher than those of p-NEOs. A positive correlation was found between the distribution of m-NEOs in albumen and their logKow (p <
0.05). Dietary intake exposure posed a higher risk of NEOs to children and females. The toxicity equivalence (TEQ) of 5-hydroxy-imidacloprid and thiacloprid-amide, in interaction with androgen and estrogen receptors, was higher compared to other NEOs. Network toxicology and molecular docking indicated that AKT1 may serve as the core target for reproductive toxicity induced by dinotefuran, cycloxaprid, and nitenpyram exposure. This study provided valuable data on the occurrence, differential distribution, and reproductive exposure risk of NEOs in eggs for the first time. These findings are instrumental for future management policies concerning the environmental behavior and health effects of NEOs.