Chronic metabolic acidosis (CMA) might result in a decrease in bone mass in vivo based on its reported in vitro inhibition of bone mineralization, bone formation or stimulation of bone resorption, but such data, in the absence of other disorders, have not been reported. CMA also results in negative nitrogen balance which might decrease skeletal muscle mass. This study analysed the net in vivo the effects of CMA's cellular and physico-chemical processes on bone turnover, trabecular and cortical bone density and bone microarchitecture using both pQCT and CT. CMA induced by NH4Cl administration (15 mEq/kg bw/ day) in intact and ovariectomized (OVX) rats resulted in stable CMA (mean {HCO3-]p = 10 mmol/L). CMA decreased osteocalcin and increased TRAP5b in intact and OVX animals. CMA decreased total volumetric bone density (vBMD) after 6 and 10 weeks (week 10: intact normal +2.1±0.9% vs. intact acidosis -3.6±1.2%, p < 0.001), an effect attributable to a decrease in cortical thickness and cortical vBMD (no significant effect on cancellous vBMD, week 10) and an increase in endosteal bone resorption (increased endosteal circumference). Trabecular bone volume (BV/TV) decreased significantly in both CMA groups at 6 and 10 weeks associated with a decrease in trabecular number. CMA significantly decreased muscle cross-sectional area in the proximal hind-limb at 6 and 10 weeks. In conclusion, chronic metabolic acidosis induces a large decrease in cortical bone mass (a prime determinant of bone fragility) in intact and OVX rats and impairs bone microarchitecture characterized by a decrease in trabecular number.