As a global public health problem, postmenopausal osteoporosis (PMOP) poses a great threat to old women's health. Bone is the target organ of PMOP, and the dynamic changes of bone marrow could affect the bone status. Kidney is the main organ regulating calcium and phosphorus homeostasis. Kidney, bone marrow and bone play crucial roles in PMOP, but the relationships of the three tissues in the disease have not been completely described. Here, metabolomics was employed to investigate the disease mechanism of PMOP from the perspectives of kidney, bone marrow and bone, and the relationships among the three tissues were also discussed. Six-month-old female Sprague-Dawley (SD) rats were randomly divided into ovariectomized (OVX) group (with bilateral ovariectomy) and sham group (with sham surgery). 13 weeks after surgery, gas chromatography–mass spectrometry (GC–MS) was performed to analyze the metabolic profiling of two groups. Multivariate statistical analysis revealed that the number of differential metabolites in kidney, bone marrow and bone between the two groups were 37, 16 and 17, respectively. The common differential metabolites of the three tissues were N-methyl-L-alanine. Kidney and bone marrow had common differential metabolites, including N-methyl-L-alanine, 2-hydroxybutyric acid, (R)-3-hydroxybutyric acid (β-hydroxybutyric acid, βHBA), urea and dodecanoic acid. There were three common differential metabolites between kidney and bone, including N-methyl-L-alanine, α-tocopherol and isofucostanol. The common differential metabolite of bone marrow and bone was N-methyl-L-alanine. Some common metabolic pathways were disturbed in multiple tissues of OVX rats, such as glycine, serine and threonine metabolism, purine metabolism, tryptophan metabolism, ubiquinone and other terpenoid-quinone biosynthesis and fatty acid biosynthesis. In conclusion, our study demonstrated that profound metabolic changes have taken place in the kidney, bone marrow and bone, involving common differential metabolites and metabolic pathways. The evaluation of differential metabolites strengthened the understanding of the kidney-bone axis and the metabolic relationships among the three tissues of OVX rats.