To characterize the dynamic mobilization of heavy metals (HM) in a crop-soil system affected by cattle manure (CM) application, soybean [Glycine max L. Merr. cv. Toyoharuka] crops were exposed in a chronological pot experiment to three CM application rates and sampled at two vegetative stages and two reproductive stages. A sequential extraction procedure for metal fractionation, soil pH, microbial activity, and plant HM uptake was determined. In non-rhizopshere soil, with CM application a liming effect was detected, and increased microbial activity was detected at the reproductive stage. CM application shifted Cd from available state to oxide-bound pool in non-rhizosphere soil; however, shifts in Cd from an oxide-bound pool to the available state were observed in rhizosphere soil. CM application stabilized the available Zn and Pb to oxide-bound Zn and organic-bound Pb in both non-rhizosphere and rhizosphere soils, and the stabilizing degree increased with higher CM application rates. The promoted Zn immobilization in the rhizosphere was due to the liming effects induced by added CM that counteracted the root-induced acidification. On the basis of a stepwise multiple regression analysis, the shift of Cd and Pb fractionation was mainly related to microbial activity. Adding manure inhibited Zn and Pb uptake but promoted Cd uptake by soybean, and a greater influence was detected at the reproductive stage, at which CM application increased the root Cd-absorbing power but did not significantly affect the Zn- and Pb-absorbing powers. In an agricultural context, long-term CM application, even at the recommended rate of 10.13 Mg ha, may cause a soybean Zn deficiency and high Pb accumulation in Haplic Fluvisols, although CM is often considered as an environmentally friendly fertilizer.