Protein undernutrition is a known factor in the pathogenesis of osteoporotic fracture in the elderly, but the mechanisms of bone loss resulting from this deficiency are still poorly understood. We investigated the effects of four isocaloric diets with varying levels of protein content (15, 7.5, 5, and 2.5% casein) on areal bone mineral density (BMD), bone ultimate strength, histomorphometry, biochemical markers of bone remodeling, plasma IGF-I, and sex hormone status in adult female rats. After 16 weeks on a 2.5% casein diet, BMD was significantly decreased at skeletal sites containing trabecular or cortical bone. Plasma IGF-I was decreased by 29–34% and no estrus sign in vaginal smear was observed. To investigate the roles of estrogen deficiency and protein undernutrition, the same protocol was used in ovariectomized (OVX) or sham-operated (SHAM) rats, pair-fed isocaloric diets containing either 15 or 2.5% casein. Trabecular BMD was decreased by either manipulation, with effects appearing to be additive. Cortical BMD was decreased only in rats on a low-protein diet. This was accompanied by an increased urinary deoxypyridinoline excretion without any change in osteocalcin levels, suggesting an uncoupling between resorption and formation. Isocaloric protein undernutrition decreased bone mineral mass and strength. This effect might be related to decreased plasma IGF-I and/or estrogen deficiency with a consequent imbalance in bone remodeling.