Oxidative defense or arsenic(As) changes exhibited byTrametes versicolorin response to toxicity under As stress remain unclear. In this study, after internal transcribed spacer identification, a wildT.versicolorHN01 strain was cultivated under 40 and 80 mg/L of AsIIIstress. The antioxidant contents by multifunctional microplate reader and the speciations of As by high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry were examined to explore the detoxification mechanisms. The results demonstrated this strain could tolerate As concentration of 80 mg/L with a bio-enrichment coefficients of 11.25. Among the four antioxidants, the activities of catalase, superoxide dismutase, and glutathione in the As-stress group at 80 mg/L improved by 1.10, 1.09, and 20.47 times that of non-stress group, respectively. The speciation results indicated that AsVwas the dominant species in the hyphae ofT.versicolorregardless of no-stress or As-stress. The detoxification mechanisms of this strain were involved alleviating the toxicity by increasing the activities of antioxidants, especially glutathione, as well as by converting AsIIIinto AsVand other less toxic As species.T.versicolorcould be used as a bio-accumulator to deal with As exposure in contaminated environments based on its extraordinary As tolerance and accumulation capacities.