SUMMARYAmmonium (NH₄⁺)‐based fertilization efficiently mitigates the adverse effects of nitrogen fertilization on the environment. However, high concentrations of soil NH₄⁺ provoke growth inhibition, partly caused by the reduction of cell enlargement and associated with modifications of cell composition, such as an increase of sugars and a decrease in organic acids. Cell expansion depends largely on the osmotic‐driven enlargement of the vacuole. However, the involvement of subcellular compartmentation in the adaptation of plants to ammonium nutrition has received little attention, until now. To investigate this, tomato (Solanum lycopersicum) plants were cultivated under nitrate and ammonium nutrition and the fourth leaf was harvested at seven developmental stages. The vacuolar expansion was monitored and metabolites and inorganic ion contents, together with intracellular pH, were determined. A data‐constrained model was constructed to estimate subcellular concentrations of major metabolites and ions. It was first validated at the three latter developmental stages by comparison with subcellular concentrations obtained experimentally using non‐aqueous fractionation. Then, the model was used to estimate the subcellular concentrations at the seven developmental stages and the net vacuolar uptake of solutes along the developmental series. Our results showed ammonium nutrition provokes an acidification of the vacuole and a reduction in the flux of solutes into the vacuoles. Overall, analysis of the subcellular compartmentation reveals a mechanism behind leaf growth inhibition under ammonium stress linked to the higher energy cost of vacuole expansion, as a result of alterations in pH, the inhibition of glycolysis routes and the depletion of organic acids.