Objective To investigate the effect of acupuncture combined with human amniotic mesenchymal stem cells (HAMCs) transplantation on the osteoporosis in ovariectomized rats. Methods Forty-five healthy female non-pregnant Wistar rats were randomly divided into sham operated group, model group and experimental group (acupuncture+HAMC transplantation), 15 rats in each group. At 7 days after the surgical wound healing, the rats received acupuncture every day and intravenous injection of HAMCs suspension (except the sham operated group) once a week for consecutive 12 weeks. The body weight was measured every week. 24 hours after the last intervention, the rats were killed and specimens were collected. Specimens of the mid-, proximal- and distal femur were taken to measure the bone mineral density using a dual energy X-ray absorptiometer. At 12 weeks after the intervention, changes of the expression of serum 25-hydroxy-vitamin D (25-HVD), C-terminal peptide of type I collagen (CTX-I), bone specific alkaline phosphatase (BAP) and human tartrate-resistant acid phosphatase 5b (TRACP 5b) were determined by ELISA, the biomechanical properties of the removed femur was measured, and the expression of transforming growth factor-β (TGF-β) in the vertebrae was assessed by RT-PCR assay. Results Body weight of rats in the sham operation group was increased gradually in accordance with the natural growth of animals, that of the model group was significantly higher than that of the sham operated group since two months after modeling, and the body weight of the experimental group was similar to that of the sham operated group. The bone mineral density and calcium content of the model group were significantly decreased compared with that of the sham operated group (P<0.05), and the bone mineral density and bone calcium content of the experimental group were significantly higher than that of the model group (P<0.05). The ELISA showed that the expressions of serum 25-HVD, CTX I, BAP, and TRAP5b in the model group were significantly lower than that of the sham operated group, and those of the experimental group were significantly higher than that of the model group (P<0.05). Measurement of biomechanical properties showed that the limit load, limit stress and elastic modulus of the femur of the sham operated group were significantly higher than that of the model group (P<0.05), and the limit load and elastic modulus of the experimental group were significantly higher than that of the sham operated group (P<0.05). T-PCR showed that the expression of TGF-β1 m-RNA in the experimental group was significantly up-regulated than that of the sham operated and model groups (P<0.05). Conclusions Acupuncture combined with human amniotic mesenchymal stem cell (HAMCs) transplantation has a synergistic effect on the treatment of osteoporosis, and can improve the conditions in ovariectomized rats.