Mushrooms have the ability to accumulate high concentrations of heavy metals, which gives them potential for use as bioremediators of environmental contamination. The Pb2+ tolerance and accumulation ability of living mycelia of Flammulina velutipes were studied in this work. Mycelial growth was inhibited when exposed to 1 mM Pb2+. The colony diameter on solid medium decreased almost 10% compared with the control. Growth decreased almost 50% when the Pb2+ concentration increased to 4 mM in the medium, with the colony diameter decreasing from 80 mm to 43.4 mm, and dry biomass production in liquid cultures decreasing from 9.23±0.55 to 4.27±0.28 g/L. Lead ions were efficiently accumulated in the mycelia. The amount of Pb2+ in the mycelia increased with increasing Pb2+ concentration in the medium, with the maximum concentration up to 707±91.4 mg/kg dry weight. We also show evidence that a large amount of the Pb2+ was adsorbed to the mycelial surface, which may indicate that an exclusion mechanism is involved in Pb tolerance. These results demonstrate that F. velutipes could be useful as a remediator of heavy metal contamination because of the characteristics of high tolerance to Pb2+ and efficient accumulation of Pb2+ ions by the mycelia.