3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test and alkaline phosphatase activity assay were employed to assess the effects of mixed trace elements including Zn(2+), Ca(2+), and Mn(2+) plus total flavonoids or icariin from Epimedium koreanum on the proliferation and differentiation of primary osteoblasts in vitro. The results indicated that icariin (0.1, 1, and 10 mu mol/L) and total flavonoids (0.06, 0.6, and 6 mu g/mL) inhibited the proliferation and promoted the differentiation of primary osteoblasts. Mixed trace elements including Zn(2+), Ca(2+), and Mn(2+) (0.1, 1, and 10 mu mol/L) inhibited the proliferation and promoted the differentiation at 0.1 and 1 mu mol/L, but inhibited the differentiation at 10 mu mol/L. The effects of mixed trace elements including Zn(2+), Ca(2+), and Mn(2+) plus total flavonoids or icariin from E. koreanum on the proliferation and differentiation of primary osteoblasts in vitro are complicated, and both synergistic and antagonistic effects are generated. The results suggest that there may be a potential cooperative action between flavonoids and trace metal elements on the proliferation and differentiation of primary osteoblasts by forming metal complexes. The combination model between flavonoids and trace metal elements is a pivotal factor for switching the biological effects from toxicity to activity, from damage to protection.