Acute myeloid leukemia (AML) is the most lethal form of leukemia. Standard anti-AML treatment remains almost unchanged for decades. Tingenone (TG) and 22-hydroxytingenone (22-HTG) are quinonemethide triterpenes found in the Amazonian plant Salacia impressifolia (Celastraceae), with cytotoxic properties in different histological types of cancer cells. In the present work, we investigated the anti-AML action mechanism of TG and 22-HTG in the AML HL-60 cell line. Both compounds exhibited potent cytotoxicity in a panel of cancer cell lines. Mechanistic studies found that TG and 22-HTG reduced cell growth and caused the externalization of phosphatidylserine, the fragmentation of internucleosomal DNA and the loss of mitochondrial transmembrane potential in HL-60 cells. In addition, pre-incubation with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, prevented TG- and 22-HTG-induced apoptosis, indicating cell death by apoptosis via a caspase-dependent pathway. The analysis of the RNA transcripts of several genes indicated the interruption of the cellular antioxidant system, including the downregulation of thioredoxin, as a target for TG and 22-HTG. The application of N-acetyl-cysteine, an antioxidant, completely prevented apoptosis induced by TG and 22-HTG, indicating activation of the apoptosis pathway mediated by oxidative stress. Moreover, TG and 22-HTG induced DNA double-strand break and phosphorylation of JNK2 (T183/Y185) and p38α (T180/Y182), and co-incubation with SP 600125 (JNK/SAPK inhibitor) and PD 169316 (p38 MAPK inhibitor) partially prevented apoptosis induced by TG and 22-HTG. Together, these data indicate that TG and 22-HTG are new candidate for anti-AML therapy targeting thioredoxin. •TG and 22-HTG induce caspase-mediated apoptotic cell death.•TG and 22-HTG target oxidative stress through downregulation of thioredoxin.•TG and 22-HTG cause DNA damage.•TG and 22-HTG cause JNK/p38-mediated apoptosis.