In order to effectively increase capacity of Cu2+ absorption by Penicillium from Cu2+-containing aqueous solution and to study the mechanisms of absorption, effects of eight pre-treatment methods on Cu2+ absorption of Penicillium janthinellum strain GXCR were compared. The results showed that the efficiency of Cu2+ absorption obviously increased through pre-treatment by homogenization, homogenization-basification (NaOH), oven dry (80oC), homogenization-salinification (NaCl), homogenization- detergent and homogenization-polarization (C2H6SO), but significantly decreased after acidification pretreatment with H2SO4. In comparison with the previous reports, the pretreatment in a homogenization-NaOH way could more efficiently enhance the Cu2+ absorption capacity of this fungus. Homogenization-basification (0.5 mol/L NaOH) increased Cu2+ biosorption by 47.95%. The Cu2+ absorption of the mycelia treated by homogenization-basification followed Langmuir isotherm equation, suggesting a surface absorption process. After four cycles of absorption-desorption, mycelia pretreated by homogenization-alkalization still had 70.82% of Cu2+ biosorption efficiency. Infrared reflectance analysis indicated that alkalization treatment made marked effects on molecular groups of C–H, C=O, and C=O in COOH on the mycelial surfaces, and -OH was a key Cu2+-binding group. It is therefore suggested that the Cu2+ absorption by the GXCR is likely to be a chemical absorption process through Cu2+ binding with -OH group on the mycelia.