The influence of metal elements on the biomedical activity of microalgal exopolysaccharides (EPS) remains underexplored. This study examined the antitumor properties of Botryococcus braunii EPS under high cobalt conditions and the role of exogenous 3-indole acetic acid (IAA) in enhancing its activity. Results showed that IAA mitigated cobalt-induced inhibition of B. braunii growth and improved its antioxidant capacity. Notably, EPS obtained from B. braunii treated with IAA under high cobalt conditions (HC-IAA-EPS) exhibited a 98.06 % inhibition of human promyelocytic leukemia cells (HL-60), significantly higher than the control (83.86 %). HC-IAA-EPS induced mitochondrial damage in HL-60 cells, evidenced by a decrease in mitochondrial transmembrane potential (observed via fluorescence microscopy) and a 1.5-fold increase in reactive oxygen species (ROS) levels compared to the control, ultimately triggering endogenous apoptosis. Proteomic analysis revealed that HC-IAA-EPS caused significant changes in apoptosis and cell cycle-related protein changes in HL-60. Gene Ontology (GO) analysis indicated enrichment in pathways such as neutrophil degranulation, Toll-like receptor (TLR) signaling, and vesicle binding complexes. This study concludes that HC-IAA-EPS inhibits HL-60 cell proliferation by inducing mitochondrial dysfunction, reducing transmembrane potential, and increasing ROS production, providing valuable insights into the antitumor potential of microalgal EPS under metal stress conditions.