Recent studies revealed that long-term intake of proton pump inhibitor (PPI) increases the risk of vertebral or hip fracture; however, the exact mechanism for this is not known. To evaluate the effect of long-term PPI therapy on bone turnover, we analyzed the signaling pathway involved in osteoclast differentiation and bone resorption/formation markers using ovariectomized rats. Six-week-old Sprague-Dawley (S-D) rats were ovariectomized, and two weeks later they were divided into four groups (group A, normal diet + placebo; group B, low calcium diet + placebo; group C, normal diet + PPI; and group D, low calcium diet + PPI). Omeprazole, at a concentration of 30 mg/kg, was administered orally for eight weeks and the rats were sacrificed when they were 16 weeks old. The relative expression levels of the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio, c-Fos, nuclear factor of activated T cells c1 (NFATc1) and osteocalcin in femoral bone marrow cells were compared, and serum C-terminal cross-linking telopeptide of type I (CTX-1) levels were determined. The relative ratio of RANKL/OPG was increased in group D, and gene expression levels of c-Fos and NFATc1 were upregulated in groups B and D, which are involved in differentiation and activation of osteoclasts. Furthermore, expression levels of osteocalcin, a bone formation marker, were decreased and levels of serum CTX-1, a bone resorption marker, were increased in group D. Taken together, a low calcium diet and PPI administration are thought to collaborate in order to alter osteoclast activity and bone resorption signaling.