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Heavy metals are present as various chemical species in soil environments. Chemical species of heavy metals determinate their behavior in soil, so they must be more important concern than total contents in treating problems about heavy metal polluted soil. Microbial activity is thought to be one of the factors that affect chemical speciation of heavy metals. Although some possible microbial mechanisms have been suggested, their effects on heavy metals have not previously been thoroughly studied. In this study, we investigated whether microorganisms contribute to the fractionation of Cu in a forest soil. Copper-enriched, sterilized forest soil samples were buried at their places of origin, and subsamples were collected for analysis 3, 10, 17, 30, 60, and 90 days after burial. Tessier’s sequential extraction was employed to analyse Cu fractionation. Until two months after burial, the added Cu remained nearly unchanged. But a dramatic change was found in soil subsamples after three months. In both the A and B horizons, the carbonate fraction of Cu increased and the residual fraction decreased. We also found that clay minerals and persistent organic matter are important binding substances of Cu in the residual fraction. The change from residual to carbonate fraction could be caused by microbial activity.