Colorectal cancer is an aggressive malignancy characterized by significant drug resistance and a complex tumor microenvironment. Nano-dihydroartemisinin liposomes demonstrate significant potential in inhibiting tumor growth and survival by targeting critical regulatory proteins involved in drug efflux and apoptosis. This study aims to investigate the mechanisms through which nano-dihydroartemisinin liposomes inhibit colorectal cancer cell proliferation and survival.The colorectal cancer cell line HCT116 and SW480 was treated with nano-dihydroartemisinin by 2D or 3D culture models. A comprehensive series of in vitro assays was conducted, including flow cytometry, CCK-8, scratch assays and morphological assessments to observe cellular changes in response to treatment. Furthermore, bioinformatics and machine learning techniques were employed to identify key molecular targets associated with the inhibitory effects of nano-dihydroartemisinin on colorectal cancer growth and survival, which were subsequently validated using RT-qPCR, immunofluorescence and omics analyses. Additionally, computational simulations were performed to assess stability of binding interactions between dihydroartemisinin and its key target proteins, further elucidating the molecular dynamics underlying its therapeutic efficacy.Cellular experiments revealed that nano-dihydroartemisinin liposomes markedly inhibited proliferation and invasive capacity of colorectal cancer, concurrently inducing significant oxidative stress within the cells. Machine learning and bioinformatics analyses identified ABCG2 and BCL2 as pivotal molecular targets involved in the therapeutic response. Subsequent RT-qPCR and immunofluorescence confirmed that treatment with nano-dihydroartemisinin liposomes led to a significant downregulation of the expression levels of these targets. Additionally, molecular dynamics simulations demonstrated that dihydroartemisinin formed stable and favorable binding interactions with the identified key protein targets.This study revealed that nano-dihydroartemisinin liposomes inhibited survival and growth of colorectal cancer by interfering with expression of ABCG2 and BCL2. Nano-dihydroartemisinin liposomes downregulated the expression of ABCG2, effectively disrupting drug efflux mechanisms to enhance intracellular drug concentrations, thereby promoting tumor cell death. Simultaneously, the downregulation of BCL2 compromised the anti-apoptotic capacity of colorectal cancer cells, leading to increased oxidative stress and metabolic dysregulation that further hindered tumor proliferation and invasiveness.Copyright © 2025 Elsevier B.V. All rights reserved.