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Selenate enhances arsenic (As) accumulation in As-hyperaccumulator Pteris vittata, but the associated molecular mechanisms are unclear. Here, we investigated the mechanisms of selenate-induced arsenic accumulation by exposing P. vittatato 50 μM arsenate (AsV50) and 1.25 (Se1.25) or 5 μM (Se5) selenate in hydroponics. After 2 weeks, plant biomass, plant As and Se contents, As speciation in plant and growth media, and important genes related to As detoxification in P. vittatawere determined. These genes included P transporters PvPht1;3and PvPht1;4(AsV uptake), arsenate reductases PvHAC1and PvHAC2(AsV reduction), and arsenite (AsIII) antiporters PvACR3and PvACR3;2(AsIII translocation) in the roots, and AsIII antiporters PvACR3;1and PvACR3;3(AsIII sequestration) in the fronds. The results show that Se1.25was more effective than Se5in increasing As accumulation in both P. vittataroots and fronds, which increased by 27 and 153% to 353 and 506 mg kg–1. The As speciation analyses show that selenate increased the AsIII levels in P. vittata, with 124–282% more AsIII being translocated into the fronds. The qPCR analyses indicate that Se1.25upregulated the gene expression of PvHAC1by 1.2-fold, and PvACR3and PvACR3;2by 1.0- to 2.5-fold in the roots, and PvACR3;1and PvACR3;3by 0.6- to 1.1-fold in the fronds under AsV50treatment. Though arsenate enhanced gene expression of P transporters PvPht1;3and PvPht1;4, selenate had little effect. Our results indicate that selenate effectively increased As accumulation in P. vittata, mostly by increasing reduction of AsV to AsIII in the roots, AsIII translocation from the roots to fronds, and AsIII sequestration into the vacuoles in the fronds. The results suggest that selenate may be used to enhance phytoremediation of As-contaminated soils using P. vittata.