A novel acridone derivative, N-(2-(dimethylamino)ethyl)-1-((3-methoxybenzyl)amino)-5- nitro-9-oxo-9,10-dihydro-acridine-4-carboxamide (8q), which was synthesized in our lab, showed potent anti-leukaemia activity against CCRF-CEM cells. Moreover, in silico predictions showed that 8q conformed to the rule of five and displayed low toxicity. However, the mechanism of anti-leukaemia action remains unclear. The aim of this research was to reveal the probable anti-leukaemia mechanism of 8q on CCRF-CEM cells. Flow cytometry assay demonstrated that 8q induced apoptosis. The expression of caspase family proteins results showed that 8q could only promote cleaved caspase-3, 7 and 9 expressions without affecting cleaved caspase-8 protein, hinting that 8q induced mitochondrial-mediated apoptosis. Further, we detected 3 indicators of mitochondrial lesions, including increased of Cyt-C release, with a decrease in MMP and ATP levels. Next, metabolomics were introduced to assist in the research of the anti-leukaemia mechanism of 8q. The metabolomics results showed that 100 nM 8q could increase the level of GSH, and decrease its oxidation products. These indicated 8q could influence the ROS, which derived by mitochondria. Then we examined the effect of 8q on intracellular ROS levels. What is particularly interesting is that 8q inhibited cell ROS stress at low concentration and stimulated ROS stress at high concentration. The pro-apoptosis mechanisms of 8q were then explored. 8q significantly decreased anti-apoptotic proteins Bcl-2 and Bcl-xL expression, whereas it up-regulated the pro-apoptotic proteins Bax, Bak, Bad, Bik and Puma expression. In addition, 8q dramatically inhibited the expression of FASN, which is related to fatty acid metabolism. Furthermore, PI3K, AKT and FOXO1 were inactivated, and the expression of total AKT was also inhibited by 8q treatment, which promoted intrinsic apoptosis. In conclusion, these findings demonstrate that 8q can induce mitochondrial lesions and promote mitochondrial-mediated pathway apoptosis by regulating the expression of Bcl-2 family proteins and inhibiting the activity of the PI3K/AKT/FOXO1 signaling pathway.