Mobile metal Cu colloids can be formed in periodically flooded paddy soils, potentially aggravating the risks to rice cultivated in these soils. Here, we investigated the formation and fate of Cu colloids in flooded soil as influenced by soil reducible sulfate and temperature. In microcosms with different initial sulfate availability (1.30, 5.34, or 7.38 mmol/kg), we found the treatments with higher sulfate concentrations showed the greater and faster release of metal colloids. Sulfate reduction resulted in the transformation of copper in the colloids from Cu(0) to CuS, and the percentage of CuS in the colloid phase increased with increasing sulfate content according to the Cu K-edge EXAFS spectra. The batch experiments incubated at 5, 25 or 35 °C proved that high temperature enhanced the microbial activity and released more Cu colloids during flooding. The colloid formation was delayed at low temperature but persisted longer in the soil, which led to greater particle average size because of slow growth and uniform agglomeration. Low temperature appeared to only influence the formation and growth but not the speciation of Cu colloids. Our results highlight the importance of soil reducible sulfate and temperature in mediating the dynamics of colloidal metals in flooded soil.