Microelements play pivotal roles for fungal/plant development and end-use properties. In this study, we examined the production and characterization of valuable sulfated polysaccharides (SPSs) with biological benefits from Antrodia cinnamomea and fine-tuning of mycelial culture conditions. Using various sulfated salts (e.g. CuSO4, FeSO4 and ZnSO4) to feed A. cinnamomea, we found that CuSO4 and ZnSO4 increased 25% and 20% of mycelium yields, respectively. We further isolated the SPSs from CuSO4, FeSO4 and ZnSO4-feeding of A. cinnamomea (called CuFSPS, FeFSPS and ZnFSPS, respectively) and found that CuSO4 and ZnSO4 significantly promoted SPS production. By contrast, FeSO4 did not change the yields of mycelium and SPS from A. cinnamomea. Characteristic studies have revealed that these sulfated salts did not significantly induce change in the sulfation and the sugar contents of SPS. However, the galactose and glucose contents in ZnFSPS were increased to the value of 249 and 1038 μmol/g, respectively. In addition, in regard to area percentages, while the major SPSs species were low-molecular-weight SPSs (<23 kDa), the sulfated salts increased the area percentages of molecular size in the range of 200-500 kDa. Anticancer function studies showed that those SPSs inhibit the cell viability 35-45% at 800 μg/ml of lung cancer A549 cells via downregulation of EGFR signaling. Our study is the first to identify the efficacy of microelements in the enhancement of mycelia yield and SPS, in which CuSO4 and ZnSO4 enhanced mycelia growth and increased the production of SPS. Our finding suggests that ZnSO4 may play roles in regulating the SPS assembling. Moreover, those SPSs derived from feeding A. cinnamomea with microelements may be useful as a potential agent for inhibition of lung cancer viability.