The functional reconstruction of large neural defects usually requires the use of peripheral nerve autografts, though these have certain limitations. As a result, interest in new alternatives for autograft development has risen. The acellular peripheral nerve graft is an alternative for peripheral nerve injury repair, but to date there is not a standardized chemical decellularization method widely accepted. The objective of this study was to propose a modified chemical protocol of decellularization of rat sciatic nerve and its recellularization in vitro with mesenchimal differentiated Schwann-like cells. After the transplantation, an evaluation of its regeneration was performed using morphological and functional tests. The study consisted of two phases; in phase 1, different concentrations and times of exposure of rat sciatic nerves to detergents were tested, to establish a modified chemical protocol for nerve decellularization. The chemical treatment with 3% triton X-100 and 4% sodium deoxycholate for 15 days allowed a complete decellularization whilst conserving the extracellular matrix of the harvested nerve. In phase 2, the decellularized and recellularized alografts were compared against autografts. The morphological analysis showed a higher positivity to specific myelin antibodies in the recellularized group compared to the autograft. There were no differences in this parameter between the control limb and the experimental limb (recellularized group). The functional analysis showed no statistical differences at week 15 in the Sciatic Function Index in the autograft group vs the other groups. This study sets the morphological and functional bases for posterior studies about nerve defects regeneration in humans.