AbstractThe anti‐apoptotic proteins, Bcl‐2 and Survivin, are consistently overexpressed in numerous human malignancies, notably in colorectal cancer. 2,4‐Di‐tert‐butylphenol (2,4‐DTBP) is a naturally occurring phenolic compound known for its diverse biological activities, including anti‐cancer properties. The mechanism behind 2,4‐DTBP‐induced inhibition of cell proliferation and apoptosis in human colorectal cancer cells, specifically regarding Bcl‐2 and Survivin, remains to be elucidated. In this study, we employed both in silico and in vitro methodologies to underpin this interaction at the molecular level. Molecular docking demonstrated a substantial binding affinity of 2,4‐DTBP towards Bcl‐2 (ΔG = −9.8 kcal/mol) and Survivin (ΔG = −5.6 kcal/mol), suggesting a potential inhibitory effect. Further, molecular dynamic simulations complemented by MM‐GBSA calculations confirmed the significant binding of 2,4‐DTBP with Bcl‐2 (dGbind = −54.85 ± 6.79 kcal/mol) and Survivin (dGbind = −32.36 ± 1.29 kcal/mol). In vitro assays using HCT116 colorectal cancer cells revealed that 2,4‐DTBP inhibited proliferation and promoted apoptosis in both a dose‐ and time‐dependent manner. Fluorescence imaging and scanning electron microscopy illustrated the classical features associated with apoptosis upon 2,4‐DTBP exposure. Cell cycle analysis through flow cytometry highlighted a G1 phase arrest and apoptosis assay demonstrated increased apoptotic cell population. Notably, western blotting results indicated a decreased expression of Bcl‐2 and Survivin post‐treatment. Considering the cytoprotective roles of Bcl‐2 and Survivin through the inhibition of mitochondrial dysfunction, our findings of disrupted mitochondrial bioenergetics, characterized by reduced ATP production and oxygen consumption, further accentuate the functional impairment of these proteins. Overall, the integration of in silico and in vitro data suggests that 2,4‐DTBP holds promise as a therapeutic agent targeting Bcl‐2 and Survivin in colorectal cancer.