Aims Both aluminum (Al) toxicity and phosphorus (P) deficiency are limiting factors of crop production on acid soils. Although Al-P interaction has been extensively studied, the results are controversial. The aim of thisstudyistoinvestigatetheeffectofinplantaPonAlinduced inhibition of root elongation in wheat (Triticum aestivum L.). Methods Roots of wheat (cv. Atlas 66) with different internal P concentrations were prepared by two methods; split-root and re-rooting in a hydroponic solution using three different P levels (0, 25 and 250 μM) to avoid direct precipitation of Al-P in the solution. Al toxicity was evaluated by root elongation inhibition and callose induction. The Al and P concentrations in the root tips were also compared among different treatments. Results Both split-root and re-rooting methods generated roots with different P concentrations in the tips when exposed to different P levels. Lower P in the root tips resulted in less Al-induced inhibition of the root elongation, less callose content and less Al accumulation, while higher root P caused a higher Al-induced inhibitionofthe root elongation, increased callose content and Al accumulation in the root tips. Furthermore, Al in the root cell sap was not altered by different P concentrations, but Al in the root cell wall was increased with increasing in planta P concentrations. Conclusions Al toxicity in wheat is associated with P in the root cell wall; lower root P enhanced Al tolerance, while higher root P aggravated Al toxicity in wheat.