Fructus Ligustri Lucidi (FLL) is a commonly prescribed herb in many kidney-tonifying Traditional Chinese Medicinal formulae for the treatment of osteoporosis. The present study aimed to identify the active fractions in FLL and to characterise its effects on Ca balance, calciotropic hormone levels as well as bone properties in mature female rats fed diets containing different levels of Ca. In the present study, 4-month-old Sprague–Dawley female rats were treated with either FLL ethanol extract (EE), ethyl acetate-soluble fraction of EE (EAF), water-soluble fraction of EE (WF) or their vehicle for 12 weeks on a medium-Ca diet (MCD, 0·6 % Ca, 0·65 % P). Then, the Sprague–Dawley female rats treated with WF or its vehicle for 12 weeks were fed diets containing different levels of dietary Ca (low-Ca diet (LCD), 0·1 % Ca, 0·65 % P; MCD; high-Ca diet (HCD), 1·2 % Ca, 0·65 % P). The results demonstrated that WF from EE but not EAF exerted a prominent effect on Ca balance by inhibiting urinary and faecal Ca excretion. WF significantly increased Ca balance in rats fed MCD or HCD with an associated increase in serum parathyroid hormone (PTH) levels. WF did not alter bone mineral density or bone mineral content of the tibia in all the rats fed with different levels of dietary Ca. In conclusion, WF was responsible for the positive actions of FLL on Ca absorption and balance. The regulation of Ca balance by WF might involve its action in stimulating PTH production in the mature female rats.