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In different cancer types, classical chemotherapy has several side effects due to the cytotoxic properties of the compounds and non-selective targeting of normal tissue. The aims of this study were to determine bioactive molecules and to investigate the genetic mechanisms of the anticancer properties of walnut special mixture, walnut milk (WM), as a potential anticancer treatment in Du145, MCF7 and TG/HAVSMC cells. The bioactive molecules of WM were determined by LC-Q-TOF analysis. After treatment with the WM, cell viability was determined using the MTT assay and apoptosis induction was observed following cell membrane staining by annexin-V/propidium-iodide using a Tali-cytometer. The gene expression studies were carried out using a qRT-PCR assay. In the WM, we quantified five hormones, eight polyphenols, quercetin and juglone. Abscisic acid (63.07 ± 18.70 μg/L), gallic acid (3887.08 ± 155.06 μg/L), quercetin (245.26 ± 34.12 μg/L) and juglone (401.52 ± 16.60 μg/L) were major components of the quantified compounds. Our results indicated that WM dramatically reduces cell viability and selectively induces caspase-dependent apoptosis in Du145 and MCF7 cells without affecting TG/HA-VSMC noncancerous cells by triggering intrinsic apoptotic signalling and increases in ROS production. Our results suggest that WM is a potential anticancer agent with selective apoptotic potential and special bioactive chemical constituents.