Sclerostin (SOST), a glycoprotein predominantly secreted by bone tissue osteocytes, is an important regulator of bone formation, and loss of SOST results in Van Buchem disease. DNA methylation regulates SOST expression in human osteocytes, although the detailed underlying mechanisms remain unknown. In this study, we compared 12 patients with bone fractures and postmenopausal osteoporosis with eight patients without postmenopausal osteoporosis to understand the mechanisms via which SOST methylation affects osteoporosis. Serum and bone SOST expression was reduced in patients with osteoporosis. Bisulfite sequencing polymerase chain reaction revealed that the methylation rate was higher in patients with osteoporosis. We identified osterix (SP7), Runt-related transcription factor 2 (RUNX2), and estrogen receptor α (ERα) as candidate transcription factors activating SOST expression. Increased SOST methylation impaired the transactivation function of SP7, RUNX2, and ERα in MG-63 cells. AzadC treatment and SOST overexpression in MG-63 cells altered cell proliferation and apoptosis. Chromatin immunoprecipitation showed that higher methylation was associated with reduced SP7, RUNX2, and ERα binding to the SOST promoter in patients with osteoporosis. Our studies provide new insight into the role of SOST methylation in osteoporosis.