β-Lapachone as a natural novel anticancer candidate is under clinical trials. Previous studies suggested that β-lapachone works by redox activation to ablate cancer cells. However, it is still unclear whether thioredoxin reductase (TrxR), one of the key redox catalytic enzymes in cells, plays a role in the pharmacological effects of β-lapachone. Herein, we present that β-lapachone kills human promyelocytic leukemia HL-60 cells with preference over other cancer cells and normal cells. The follow-up studies demonstrate that β-lapachone induces the HL-60 cell apoptosis through inhibition of TrxR and further elevation of oxidative stress. Overexpression of the TrxR alleviates the efficiency of β-lapachone while knockdown of the enzyme increases the β-lapachone cytotoxicity, scientifically underpinning the correlation of the observed biological behaviors of β-lapachone to TrxR inhibition. The disclosure of the novel action mechanism of β-lapachone sheds light on understanding its capacity in interfering with cellular redox signaling and supports β-lapachone as an anticancer drug candidate.