AbstractPerturbations in autophagy, apoptosis and differentiation have greatly affected the progression and therapy of acute myeloid leukaemia (AML). The role of X‐linked inhibitor of apoptosis (XIAP)‐related autophagy remains unclear in AML therapeutics. Here, we found that XIAP was highly expressed and associated with poor overall survival in patients with AML. Furthermore, pharmacologic inhibition of XIAP using birinapant or XIAP knockdown via siRNA impaired the proliferation and clonogenic capacity by inducing autophagy and apoptosis in AML cells. Intriguingly, birinapant‐induced cell death was aggravated in combination with ATG5 siRNA or an autophagy inhibitor spautin‐1, suggesting that autophagy may be a pro‐survival signalling. Spautin‐1 further enhanced the ROS level and myeloid differentiation in THP‐1 cells treated with birinapant. The mechanism analysis showed that XIAP interacted with MDM2 and p53, and XIAP inhibition notably downregulated p53, substantially increased the AMPKα1 phosphorylation and downregulated the mTOR phosphorylation. Combined treatment using birinapant and chloroquine significantly retarded AML progression in both a subcutaneous xenograft model injected with HEL cells and an orthotopic xenograft model injected intravenously with C1498 cells. Collectively, our data suggested that XIAP inhibition can induce autophagy, apoptosis and differentiation, and combined inhibition of XIAP and autophagy may be a promising therapeutic strategy for AML.