Osteoporosis is described as an age-associated impairment of bone formation. microRNA (miR)-29b-3p was thought to be linked to osteoblast differentiation; however, the underlying molecular pathways are yet unknown. The study's goal was to look into the involvement of miR-29b-3p in osteoporosis and the pathophysiological mechanisms. A murine model of estrogen deficiency-induced bone loss was established to simulate postmenopausal osteoporosis. Reverse transcription-quantitative PCR (RT-qPCR) was performed to assess the level of miR-29b-3p of bone tissue. Additionally, miR-29b-3p/sirtuin-1 (SIRT1)/peroxisome proliferator-activated receptor gamma (PPAR gamma) axis in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was examined. Osteogenesis-related markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2), were assessed at protein and molecular levels. ALP staining and Alizarin Red staining were used to detect ALP activity and calcium deposition. The ovariectomy group was shown to express miR-29b-3p at higher levels in vitro, and miR-29b-3p mimics suppressed osteogenic differentiation and protein/mRNA expression levels of osteogenesis-related markers in vivo. SIRT1 was identified as a target of miR-29b-3p using luciferase reporter assays. Overexpression of SIRT1 reduced the inhibition of osteogenic differentiation by miR-29b-3p. Rosiglitazone, an activator of PPAR gamma signaling, was able to reverse the downregulation of the osteogenic differentiation of BMSCs and the protein expression of PPAR gamma caused by miR-29b-3p inhibitors. The results revealed that osteogenesis was suppressed by miR-29b-3p, which blocks the SIRT1/PPAR gamma axis. These results suggested that postmenopausal osteoporosis could be treated by targeting miR-29b-3p SIRT1/PPAR gamma.