A two-year field experiment was set up in Central-Eastern Poland (Siedlce Upland) to study the influence of fertilisation with spent substrate after the cultivation of white mushrooms (Agaricus bisporus) on the content of nickel and its overall quantitative participation in separated fractions in the humus horizon of Stagnic Luvisol under agricultural use. The experimental treatments were as follows: control (without fertilisation); with NPK mineral fertilisation; fertilised with swine manure; fertilised with swine manure + NPK; fertilised with spent mushroom substrate; fertilised with spent mushroom substrate + NPK. Sequential fractionation of nickel was conducted according to the BCR procedure. The following nickel fractions were separated: F1– exchangeable, F2 – reducible, F3 – oxidisable, F4 – residual. In the humus horizon of the particular experimental treatments the sequential fractionation of nickel revealed diversified content of this metal in the separated fractions and their varied share in the total content of nickel. After the first year of cultivation, the application of spent mushroom substrate to the soil, both alone and with NPK, caused an increase in the share of nickel in the exchangeable and oxidisable fractions relative to the control. After the second year of cultivation the substrate used caused a potential increase in the bioavailability and mobility of the metal in the soil, with simultaneous decrease of its share in the oxidisable fraction, bound with organic matter. The average percentage shares of nickel fractions after two years of cultivation were in the following order of decreasing values: F4 > F3 > F1 > F2.