The promising unicellular colonial Botryococcus braunii can secrete large amounts of bioactive exopolysaccharides. Our previous study revealed Botryococcus could tolerate high-concentrations of metallic cobalt, and extracellular hydrocarbons increased simultaneously. However, the characteristics of the extracellular polysaccharides (EPS) closely related to extracellular hydrocarbons is still unclear. This research focuses on the effects of cobalt exposure on the production, structure, and bioactivity of exopolysaccharides from colonial Botryococcus, aiming to provide a theoretical clue on the potential contribution of EPS to the cobalt tolerance ability in this alga for cobalt bioremediation from the perspective of structure-bioactivity relationship.The results showed that high cobalt treatment (4.5 mg/L) exerted little influence on the growth of the alga, or the total carbohydrate content obtained, compared to the algae without cobalt treatment. EPSs of B. braunii SAG 30.81, under normal condition (EPS-Co) was mainly composed of glucose, mannose, fucose, galactose, rhamnose, and xylose, but also with the presence of sulfate substitutions, which was unique among the EPSs previously reported in this alga. Cobalt treatment resulted in a remarkable decline of rhamnose, and an increase of ribose and sulfate content, with more esterified uronic acids in the EPS (EPS + Co). EPS + Co, overall, had a larger but more compact and branched, spherical-like EPSs, with more ordered (helix) chains relative to EPS-Co. EPS + Co exhibited stronger antioxidant and antibacterial activity than EPS-Co, which could be attributed to the changed structural characteristics, such as the decrease of Rha, Fuc, and Gal and an increase of sulfate substitutions, uronic acids and Ara, more branched structure, and more ordered conformation. The enhanced antioxidant activity could be contributed to the alga tolerance to cobalt exposure, which was benefitted by cobalt bioremediation.