Currently, lead (Pb) has become a severe environmental pollutant and fungi hold a promising potential for the remediation of Pb-containing wastewater. The present study showed that Penicillium polonicum was able to tolerate 4 mmol/L Pb(II), and remove 90.3% of them in 12 days through three mechanisms: extracellular immobilization, cell wall adsorption, and intracellular bioaccumulation. In this paper. the three mechanisms were studied by Raman, X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The results indicated that Pb(II) was immobilized as lead oxalate outside the fungal cell, bound with phosphate, nitro, halide, hydroxyl, amino, and carboxyl groups on the cell wall, precipitated as pyromorphite [Pb5(PO4)3Cl] on the cell wall, and reduced to Pb(0) inside the cell. These combined results provide a basis for additionally understanding the mechanisms of Pb(II) removal by P. polonicum and developing remediation strategies using this fungus for lead-polluted water.