Microgravity induces significant and progressive bone loss in both humans and animals. This is the consequence of disturbed bone remodeling. We performed a bed rest experiment to simulate microgravity and tried to clarify bone metabolism by measuring biochemical markers of bone turnover. Six healthy volunteers participated in 120 days of bed rest. The parameters of calcium homeostasis, calcitropic hormones, and biochemical markers of bone turnover were examined. After ambulatory control evaluation, all subjects underwent 120 days of bed rest. Metabolic evaluation was performed in a baseline period, and on days 7, 16, 50, 72, 92, and 108 during bed rest, and on days 10 and 25 during a recovery period. Bed rest induced an increase in urinary calcium (Ca) excretion and serum Ca and bone resorption markers. Urine pyridinoline, deoxypyridinoline, and type I collagen cross-linked N-telopeptide increased more rapidly than urinary Ca excretion and serum Ca. Tartrate-resistant acid phosphatase (TRAP) increased even in the recovery period. Carboxy-terminal propeptide of type I collagen, a bone formation marker, significantly decreased on days 50, 92, and 108 of bed rest. These changes of biochemical markers of bone metabolism, except for TRAP, rapidly returned toward control levels in the recovery period. Immunoreactive parathyroid hormone showed a modest decrease during bed rest and a significant increase in the recovery period. Insulin-like growth factor I (IGF-I) and its binding protein, insulin-like growth factor binding protein-3, increased during bed rest, indicating the possibility of resistance to IGF-I in bones under reduced mechanical stress and strain. Bone loss from unloading results from the combination of acceleration of bone resorption and subsequent retardation of bone formation.