Applying sewage sludge or animal manure onto agricultural land can result in estrogen pollution, which increases the risk of human exposure to steroid estrogens (SEs) via the food chain. However, the uptake and accumulation mechanism of SEs by plants is still unclear. In this study, the uptake, accumulation, and translocation of 17β-E2, a representative SE, were investigated through a series of wheat hydroponic experiments. Various inhibitors were applied to explore the uptake pathways of 17β-E2 by wheat. In addition, the effects of exposure concentrations, coexisting 17α-ethynylestradiol (EE2) and plant properties on the uptake of 17β-E2 were examined. The results indicated that the accumulation of 17β-E2 in wheat roots mainly resulted from adsorption and active uptake that involved aquaporins and anion channels transport. The chlorophyll and protein contents of plants were positively correlated with the uptake of 17β-E2, whereas competitive inhibition occurred when 17β-E2 and EE2 coexisted in the same solution. Nevertheless, the results of a split-root experiment showed that 17β-E2 absorbed by wheat could further migrate in plant via long-distance transport and ultimately was discharged from plants, suggesting that 17β-E2 was still at risk of being released even though it had been absorbed by plants. These results could provide valuable insights into the risk assessment and risk control of the uptake of SEs by plants.