The gradually accumulation of nutrients in organic substrates during growing season may have adverse eﬀects on plant development. This study was conducted to determine the mode of nutrient accumulation and alteration at molecular level induced by the excess nutrient stress in tomato. The ICP-MS was used to analyze mineral ions in root zone solution and nutrients uptake by tomato plants; and iTRAQ-based proteomic analysis was conducted to identify proteome changes in tomato roots. Coir and peat-vermiculite were used in substrate treated experiments. Results showed that the content of K+ was higher in coir than in peat-vermiculite whereas it was the opposite for Ca2+. The concentrations of NO3−, SO42- and Mg2+ were signiﬁcantly higher in peat-vermiculite and coir substrates than in water culture. The peat-vermiculite substrates generally enhanced Ca uptake but reduced P uptake by plants, when compared to both coir and water culture. Compared to water culture, functional annotation analysis of the root proteome revealed that the excess nutrient accumulation induced complex proteomic alterations involved in mineral ion binding and transport. A total of 358 diﬀerentially abundant proteins (DAPS) were identiﬁed, including 11 mineral ion binding and transport related proteins, such as calmodulin-like protein and nitrate transporter 3.2 under peat-vermiculite and coir cultivations. RT-qPCR was used to validate nine genes encoding DAPS. We believe that these indicators will contribute to a better control of soilless culture systems and a waste reduction in production of tomatoes.