The human amniotic membrane (hAM) may be helpful as a support for bone regeneration. To assess its potential for bone repair, a wide heterogeneity of preservation methods of hAM has been studied. The objectives of this study were: i) to assess bone regeneration potential of fresh versus cryopreserved hAM, and ii) to characterize hAM depending on four preservation methods. hAM was used either fresh (F-hAM), cryopreserved (C-hAM), lyophilized (L-hAM) or decellularized and lyophilized (DL-hAM). First, critical calvarial bone defects were performed in mice. Defects remained empty or were covered by F-hAM or C-hAM. Then, the cytotoxicity of the four preservation methods of hAM was assessed in vitro on human bone marrow mesenchymal stem cells (hBMSCs), and, their biocompatibility was evaluated in vivo in a rat subcutaneous model. X-Rays analysis showed that no calvarial defect was regenerated ad integrum. Bone regeneration was slightly enhanced by C-hAM. In vitro, the decellularization and the lyophilization process did not confer any cytotoxicity of the tissue compared to other preservation methods. In vivo, L-hAM and DL-hAM were easier to handle. Histological analysis of explanted samples from the rat indicated a slight to moderate inflammatory reaction with hAM. One month after surgery, a complete resorption of F-hAM and C-hAM implants occured, whereas L-hAM and DL-hAM were still observed. C-hAM has a limited potential for GBR. L-hAM and DL-hAM are biocompatible without cytotoxic effects. These preservation methods should be suitable in the field of bone regeneration.