This study was to determine the effect of soil amendments on the fractionation of selenium (Se) using incubation experiments under simulated upland and flooded conditions. The treatments were as follows: 1) control [soil + sodium selenite (Na2SeO3) (1 mg Se kg‐1)]; 2) control + calcium carbonate (CaCO3) (5 g kg‐1); 3) control + alfalfa (40 g kg‐1); and 4) control + CaCO3(5 g kg‐1) + alfalfa (40 g kg‐1). After a 90‐day incubation, soil was sampled and fractionated into five fractions: 1) potassium sulfate (K2SO4)‐soluble fraction (available to plants); 2) potassium dihydrogen phosphate (KH2PO4)‐exchangeable fraction (potentially available); 3) ammonium hydroxide (NH3H2O)‐soluble fraction (potentially available); 4) hydrochloric acid (HCl)‐extractable fraction (unavailable); and 5) residual fraction (unavailable). Compared with the control, CaCO3increased the K2SO4fraction at the expense of the NH3H2O fraction. Alfalfa increased both the K2SO4and residual fractions but reduced the KH2PO4and NH3H2O fractions. The CaCO3‐alfalfa treatment had a similar effect to the alfalfa treatment alone. The comparison between the upland and flooded conditions showed that the flooded condition generally increased the residual fraction and decreased the potentially‐available fractions. In general, CaCO3was a better amendment because it not only increased the available fraction but also maintained the potentially available fractions at a high level. The application of Na2SeO3and use of appropriate soil amendments can improve Se availability in soil.