Decitabine (DAC) is a well-known DNA methyltransferase inhibitor, which has been widely used for the treatment of acute myeloid leukemia (AML). However, in addition to hypomethylation, DAC in AML is also involved in cell metabolism, apoptosis, and immunity. The TP53-induced glycolysis and apoptosis regulator (TIGAR) functions to inhabit glycolysis and protect cancer cells from reactive oxygen species- (ROS-) associated apoptosis. Our previous study revealed that TIGAR is highly expressed in myeloid leukemia cell lines and AML primary cells and associated with poor prognosis in adult patients with cytogenetically normal AML. In the present study, it was found that in a time- and concentration-dependent manner, DAC downregulates the TIGAR expression, induces ROS production, and promotes apoptosis in HL-60 and K562 cells. However, blocking the glycolytic pathway partially reversed the combined effects of DAC and TIGAR knockdown on apoptosis, ROS production, and cell cycle arrest, indicating that DAC induced apoptosis through the glycolytic pathway. Furthermore, TIGAR also has a negative impact on autophagy, while DAC treatment upregulates autophagy-related proteins LC3, Beclin-1, ATG3, and ATG-5, downregulates p62, and promotes the formation of autophagosomes, indicating that DAC may activate autophagy by downregulating TIGAR. Taken together, DAC plays an unmethylated role in inducing apoptosis and activating autophagy in myeloid leukemia by downregulating TIGAR.