We investigated the effects of earthworm activity on the bioavailability of Cu in soil. The bioavailable fraction was estimated using sequential extraction, and the results of diethylenetriaminepentaacetic acid (DTPA) extraction were analyzed for comparison. Changes in the Cu fraction were compared in Cu-spiked soil (high bioavailability) and long-term polluted field soil (low bioavailability) with approximately equivalent total Cu concentrations. Earthworm activity decreased the bioavailable fraction in the Cu-spiked soil, where earthworm body Cu concentrations did not affect the bioavailable fraction. Soil pH was not a factor in the bioavailability differences between soils with and without earthworms in this study. The bioavailable fraction appears to be more heavily affected by biological and physical mechanisms than by soil pH. The two extraction methods showed different trends; the bioavailable fraction method was better than DTPA extraction, because the former gives clear insight into the aging process of Cu in soil.