Dietary intake of plant-based foods is a major contribution to the total exposure of polycyclic aromatic hydrocarbons (PAHs). However, the mechanisms underlying PAH uptake by roots remain poorly understood. This is the first study, to our knowledge, to reveal cytoplasmic pH change and regulation in response to PAH uptake by wheat roots. An initial drop of cytoplasmic pH, which is concentration-dependent upon exposure to phenanthrene (a model PAH), was followed by a slow recovery, indicating the operation of a powerful cytoplasmic pH regulating system. Intracellular buffers are prevalent and act in the first few minutes of acidification. Phenanthrene activates plasmalemma and tonoplast H(+) pump. Cytolasmic acidification is also accompanied by vacuolar acidification. In addition, phenanthrene decreases the activity of phosphoenolpyruvate carboxylase and malate concentration. Moreover, phenanthrene stimulates nitrate reductase. Therefore, it is concluded that phenanthrene uptake induces cytoplasmic acidification, and cytoplasmic pH recovery is achieved via physicochemical buffering, proton transport outside cytoplasm into apoplast and vacuole, and malate decarboxylation along with nitrate reduction. Our results provide a novel insight into PAH uptake by wheat roots, which is relevant to strategies for reducing PAH accumulation in wheat for food safety and improving phytoremediation of PAH-contaminated soils or water by agronomic practices.