Bone nonunion remains a challenge during the treatment of bone defect accompanied with infection. Epimedii herba is a widely used medicine in clinic to enhance bone healing. Epimedin C and icariin are the major flavonoid glycosides from Epimedii herba. Although the effects of these compounds have been reported, their different absorption and utilisation by osteoblasts remain unclear. In the present study, lipopolysaccharide (LPS)-induced osteoblast was adopted as the model cell to evaluate the effects of epimedin C and icariin. The intracellular and extracellular drug concentrations within 24 h were assayed by pipette tip solid-phase extraction and high-performance liquid chromatography, respectively. MTT, alkaline phosphatase (ALP) and calcified nodule staining were performed to identify and evaluate the effects of epimedin C and icariin on LPS-induced osteoblasts. The regulatory roles of epimedin C and icariin in the bone morphogenetic protein-2 (BMP-2)/Runt-related transcription factor 2 (Runx2) signalling pathway were investigated. The results revealed that epimedin C and icariin were not efficiently absorbed by LPS-induced osteoblasts. Nevertheless, they still had high utilisation efficiency after entering the cells. ALP activity, mineralisation and osteoblasts proliferation were enhanced by a high concentration of epimedin C and icariin. The suppressed expression of BMP-2 and Runx2 in LPS-induced osteoblasts was up-regulated significantly after treatment with epimedin C and icariin. These findings firstly illustrated the behavior of epimedin C and icariin from Epimedii herba on LPS-induced osteoblasts and the regulatory property on the expression of key genes and proteins of the BMP-2/Runx2 signalling pathway, which might be helpful for better understanding flavonoids’ mechanism to enhance bone repair and improving their future application.