In this paper, the accumulation and morphological transformation of calcium (Ca) and magnesium (Mg) in typical fluvo-aquic soil under different long-term (1989–2009) fertilization treatments were studied and the roles of Ca²⁺, Mg²⁺ and carbonate to soil buffering ability and organic matter accumulation were explored. 7 fertilization treatments were designed in the field experiment: 1) CK (non-fertilized control), 2) OM (organic manure), 3) OM + NPK (50% organic manure + 50% mineral fertilizer), 4) NPK, 5) NP, 6) PK and 7) NK. The results obtained by volume method and the modified BCR-sequential extraction method showed that the contents of Ca and Mg in topsoil (0-20 cm) accumulated continuously, and were in the order of NK < OM < OM+NKP < NPK < NP < PK < CK, which was in accordance with the contents of soil crystal lattice-bound Ca and Mg and carbonate-bound Ca and Mg, while contrary to the contents of metal oxide-bound and organo-bound Ca and Mg. pH, contents of Ca and Mg were lower under OM treatment, but OM treatment had little impact on the content of soil carbonate. CaCO₃ is the main carbonates in soil, followed by MgCO₃, they are bound to soil organic matter to form a complex acid-base buffering system in soil. Compared with Mg²⁺, Ca²⁺ are easier to bind to the metallic oxide, whereas are weaker to be bound to soil organic matter, which may inhibit the formation of soil organo-mineral complexes.