Arsenic (As) contamination in soils has posed a severe threat to safe crop production.  The previous studies showed the antagonism between phosphorus (P) and As in plant growth and As uptake, while the mechanisms of alleviating As toxicity by P is not completely clear.  Due to the limiting P condition, it is imperative to understand how low P addition can be used to suppress arsenate As (V) uptake and the subsequent mechanisms involved.  Thus in this study we investigated the effect of P addition on As uptake, anti-oxidative enzyme activity, and anti-oxidant content, and the relative expression of transport, defense, and detoxification genes using two barley genotypes differing in As toxicity tolerance.  P addition significantly reduced As concentration in plant tissues, and caused the great changes in activities of catalase and superoxide dismutase, glutathione content, and the relative expression of examined genes when the plants of the two barley genotypes were exposed to 100 µmol L^–1 As, with ZDB160 (As-tolerant) being much more affected than ZDB475 (As-sensitive).  The current results show that P addition can alleviate As toxicity by regulating the expression of As transport, defense, and detoxification genes to a greater extent in As tolerance of barley, suggesting the possibility of controlling As uptake and toxicity by applying low amount of P fertilizers in the As-contaminated soils.