Abstract: In order to reveal the biochemical mechanism and utilization of Se (Ⅵ) in roots of flue-cured tobacco, hydroponic experiments were conducted to study the kinetics of Se (Ⅵ) absorption in cultivars K326 and Yunyan87 under different pH, accompanying ions and inhibitor conditions through ion depletion. The results showed that compared with K326, Yunyan87 belonged to a high I_max (maximum uptake rate) and low K_m (dissociation constant) tobacco cultivars, and had a higher Se (Ⅵ) nutrient efficiency. Under weak acidic (pH=6) to neutral (pH=7) conditions, it was more conducive to the absorption of Se (Ⅵ), while a strong acid environment (pH=5) reduced the absorption efficiency of Se (Ⅵ). The presence of accompanying ions SO₄^2- and PO₄^3- inhibited the absorption and accumulation of Se (Ⅵ) in the roots and reduced the affinity of the tobacco to Se (Ⅵ), with SO₄^2- being a stronger effect. The addition of respiratory inhibitor CCCP and channel inhibitor LaCl₃ significantly inhibited the absorption of roots to Se (Ⅵ), and CCCP had a greater influence than LaCl₃.