The significance of metal ion supplementation in the fermentation medium on the structure and antitumor activity of Tuber polysaccharides was systematically studied in the submerged fermentation of Tuber melanosporum. The lowest weight-average molecular weight (M-w) (i.e., 115.3 x 10(4) g/mol) of intracellular polysaccharides (IPS) was obtained when Mg2+ and K+ was added in the fermentation medium. The IPS with the lower Mw exhibited a higher inhibition ratio against S-180 tumor cells. The compact conformation of extracellular polysaccharides (EPS) was formed when only K+ was supplied in the fermentation medium. Interestingly, EPS with compact conformation exhibited a higher inhibition ratio (i.e., 59.2%) than EPS with branched polymer chain (i.e., 9.2%) against A549 tumor cells. The highest inhibition ratio for EPS with alpha-glycosidic linkages against the tumor cell line HepG2 reached 32.2% when Mg2+ or K+ was supplied in the fermentation medium. The addition of metal ion Mg2+, K+, and their combination to the fermentation medium is a vital factor affecting the structures of Tuber polysaccharides, which further determine their anti-tumor activities. The information obtained in this work will be useful for the efficient and directed production of polysaccharides with anti-tumor activities by the submerged fermentation of edible fungi mycelium. (C) 2014 Elsevier Ltd. All rights reserved.