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Phosphate (PO4 (3-) ) has been reported to suppress arsenate (As(v) ) uptake in plants. However, its effects on controlling the availability of As(v) in tobacco genotypes with different arsenic (As) tolerances has not been fully explored. In the present study, the effects of PO4 (3-) on As(v) uptake were investigated in a hydroponic culture using 2 tobacco (Nicotiana tabacum) genotypes (ZY90 and FSMY) that differed in As(v) tolerance. A total of 9 treatment combinations comprising As(v) treatments of 0 µM, 10 µM, and 100 µM and PO4 (3-) treatments of 0 µM, 50 µM, and 500 µM were used. The results showed that ZY90 had greater reductions in leaf photosynthetic parameters, root and shoot dry weight, length, and nutrient content than did FSMY when exposed to As(v) stress. The addition of 500 µM external PO4 (3-) significantly suppressed As(v) (100 µM) uptake in both FSMY and ZY90, with the effect being more pronounced in FSMY. Greater PO4 (3-) uptake in plants significantly reduced the influx of As(v) , causing an increase in photosynthesis and nutrient uptake. Phosphate supply increased superoxide dismutase activity, catalase activity, and malondialdehyde content. The present study showed that PO4 (3-) is an effective competitive inhibitor of As(v) , and it can be effectively used to control As(v) accumulation in tobacco plants.