Two wheat (Triticum aestivum L.) cultivars differing in As-tolerance were used to investigate the effects of phosphorus (P) concentration and nutrient solution pH on As(V) toxicity and As(V) uptake kinetics, and to illustrate the mechanism of As(V) tolerance in wheat seedlings. Low pH and low phosphate concentration enhanced wheat uptake of As, resulting in high As toxicity. The As(V)-tolerant cultivar MM45 exhibited higher relative root elongation than non-tolerant cultivar HM29 in all treatments, except that no genotypic difference was recorded for the solution P at 100 µmol L^–1 or greater. Wheat seedling As(V) tolerance was positively correlated with P concentration in roots and shoots. In short-term (30 min) As(V)-uptake kinetics experiments, the maximum influx rate (V_max) of As(V) increased with decreasing solution pH (from 7.0 to 6.0). Compared with HM29, although MM45 had lower V_max , its Michaelis–Menten constant (K_m) did not exceed that of HM29 in any of the treatments. The V_max values of both cultivars were not significantly affected by phosphate treatments, except for HM29 which had significantly higher V_max value in the presence of phosphate at pH 7.0. The K_m values of the two cultivars increased by 9- to 20-fold when phosphate was present as opposed to absent from the uptake solution. This study showed that the V_max values are mainly increased by high pH and As(V) uptake K_m is mainly increased by phosphate presence. Decreased As(V) influx rates during early stages and increased P concentration in plant tissues are associated with increased As tolerance in wheat seedlings.