PAHs affect the uptake of heavy metal by plants. The uptake pathway, distribution and detoxification of nickel (Ni) in Agropyron cristatum L. (A. cristatum) were investigated in the presence of pyrene in this study. Most of Ni was adsorbed on the cell wall in the insoluble phosphate (57.31-72.18%) form and pectate and protein integrated (38.27-38.98%) form. Ni was transferred to the organelle (from 37.84% to 40.52%) in the presence of pyrene. The concentration of Ni in A. cristatum decreased by 27.42%; it was affected by the ATP production inhibitor and 29.49% by the P-type ATPase inhibitor. The results indicated that the uptake of Ni related closely to the synthesis and decomposition of ATP and was an active uptake process. Contents of phytochelatins (PCs) in A. cristatum in Ni contaminated soils increased by 19.97%, and an additional 4.13% increase occurred in the presence of pyrene when compared to single Ni contamination. The content of malic acid in A. cristatum was the highest for 262.78 mg g^-1 in shoots and 46.81 mg g^-1 in roots with Ni contamination. Besides, acetic acid in shoots and roots increased by 40.25% and 102.63% with Ni contamination, and by 61.59% and 185.71% with Ni-pyrene co-contamination. This study preliminarily explored the inhibitory mechanism of pyrene on plant uptake of Ni.