This study aims to evaluate the synergistic effects of doxorubicin (DOX) and the natural phenolic alkaloid avenanthramide C (AVC) on enhancing apoptosis in MCF-7 breast cancer (BC) cells, with a specific focus on the expression of mitochondrial proteins voltage-dependent anion channel 2 (VDAC2) and mitochondrial carrier homolog 1 (MTCH1) involved in apoptosis pathways.MCF-7 cells were treated with various concentrations of DOX and AVC. The 50% inhibitory concentration (IC50) of DOX combined with AVC was evaluated in the MCF-7 cells using an MTT assay, while gene expression levels of Ki-67, MTCH1, and VDAC2 were assessed through real-time polymerase chain reaction. Apoptotic rates were measured using flow cytometry.DOX and AVC combination reduced the IC50 value by 2.1-fold compared to DOX alone, indicating enhanced cytotoxicity. Co-treatment significantly downregulated Ki-67 expression and increased apoptosis by 76% in cells treated with 3 μM DOX and 160 μM AVC. Gene expression analysis revealed a 4.8-fold increase in MTCH1 and a 15-fold increase in VDAC2 compared to DOX treatment alone.The DOX-AVC combination demonstrated a potent synergistic effect, enhancing apoptosis in BC cells by modulating mitochondrial pathways. This approach may provide a promising strategy for reducing chemotherapy side effects in BC treatment. Further studies in pre-clinical models are warranted to explore its therapeutic potential.