Iso-suillin, an isomer of suillin that belongs to the prenylphenol class of fungal derivatives, was isolated from petroleum ether extracts of Suillus flavus. The IC50 value of iso-suillin in K562 cells was 0.87 μM, which was lower than the positive control cisplatin (19.33 μM). Iso-suillin-treated K562 cells exhibited an increased rate of apoptosis, mitochondrial membrane potential (MMP) depolarization, and G0/G1 arrest. Western blot analysis revealed that these cells displayed significantly upregulated expression of several apoptosis-related proteins, including cytochrome c, caspase 9, FADD (Fas-associating protein with a novel death domain), caspase 8, caspase 3, and Bax. Moreover, the expression of two anti-apoptosis proteins, NF-κB and Bcl-2, was downregulated. Inhibitors of caspase 9 and caspase 8 protected the K562 cells from apoptosis. Taken together, our results suggest that iso-suillin induces K562 apoptosis through the mitochondrial and death receptor pathways and that iso-suillin may represent a candidate anti-leukemia treatment.