Our previous study demonstrated that 60 % ethanol crude extract of Sambucus williamsii HANCE (SWH) improved bone mass, bone strength and bone micro-structure in both ovariectomised (OVX) rats and mice. The present study aims to identify the bioactive fractions and ingredients in SWH that account for its osteoprotective effects. Bilateral sham-operated mice acted as controls. OVX C57BL/6J mice, aged 12 weeks, were orally administrated daily with vehicle or 17β-oestradiol (3·2 mg/kg), SWH (60 % ethanol crude extract; 1·0 g/kg), SWA (water eluate; 0·570 g/kg), SWB (30 % ethanol eluate; 0·128 g/kg) or SWC (50 and 95 % ethanol eluates; 0·189 g/kg) for 12 weeks. The effects of the different fractions on bone properties in the OVX mice model were studied. In addition, their effects on osteoblast proliferation and differentiation were evaluated in UMR 106 cells. SWC significantly restored bone mineral density and improved bone size and bone content parameters in the femur and tibia as well as increased biomechanical strength at the tibia diaphysis in OVX mice. Similarly, SWC was the most potent fraction in stimulating cell proliferation and differentiation in UMR 106 cells. Also, SWC did not alter uterus weight in OVX mice. Nine major peaks, seven lignans and two phenolic acids, in the HPLC fingerprint of the SWC fraction were identified, isolated and characterised. In conclusion, the present study demonstrated that SWC was the most potent fraction in SWH that exerted anti-osteoporotic effects. Furthermore, lignans might be the potential bioactive components in SWC.