9,11-Dehydroergosterol peroxide [9(11)-DHEP] is an important steroid from medicinal mushroom, which has been reported to exert antitumor activity in several tumor types. However, the role of 9(11)‑DHEP toward the malignant melanoma cells has not been investigated. In the present study, the steroid from Ganoderma lucidum was purified on a submerged culture, and its antitumor mechanisms on A375 human malignant melanoma cells was investigated by MTT, flow cytometry and western blotting. The studies demonstrated that apoptotic mechanisms of the steroid were caspase‑dependent and mediated via the mitochondrial pathway. The steroid did not cause significant changes in the expression levels of B‑cell lymphoma 2 (Bcl‑2) family proteins, Bcl‑2‑like protein 11, p53 upregulated modulator of apoptosis, Bcl‑2‑associated X protein, BH3 interacting‑domain death agonist, Bcl‑2‑associated death promoter and Bcl‑2, but it significantly downregulated induced myeloid leukemia cell differentiation protein Mcl‑1 (Mcl‑1) in melanoma cells, suggesting the key role of Mcl‑1 in regulating apoptosis of melanoma cells induced by the steroid. These properties of 9(11)‑DHEP advocate its usage as supplements in human malignant melanoma chemoprevention. The present study also suggests that mycelium of G. lucidum has a potential for producing bioactive substances and extracts with applications in medicine.