Nowadays, researchers pay a vast deal of attention to neural tissue regeneration due to its tremendous effect on the patient's life. There are many strategies, from using conventional autologous nerve grafts to the newly developed methods for reconstructing damaged nerves. Among the various therapeutic methods, incorporating highly potent biomolecules and growth factors, the damaged nerve site would promote nerve regeneration. The aim was to examine the efficiency of a mesenchymal stem cell condition medium (MSC-CM) loaded on a 3D-polycaprolactone (PCL) scaffold as a nerve conduit in an axotomy rat model. Twenty-four mature male rats were classified into four groups: controls (the animals of this group were intact), axotomy (10 mm piece of the nerve was removed), axotomy (10-mm piece of the nerve was removed) + scaffold, and axotomy (10-mm piece of the nerve was removed) + MSC-CM-loaded scaffold. We followed up nerve motor function using a sciatic function index and electromyography activity of the gastrocnemius muscle. At 12 weeks post axotomy, sciatic nerve and dorsal root ganglion specimens and L4 and L5 spinal cord segments were separated from the rats and were analyzed by stereological, immunohistochemistry, and RT-PCR procedures. The rats of the axotomy group presented the expected gross locomotor deficit. Stereological parameters, immunohistochemistry of GFAP, and gene expression of S100, NGF, and BDNF were significantly enhanced in the CM-loaded scaffold group compared with the axotomy group. The most observed similarity was noted between the results of the control group and the CM-loaded scaffold group. Our results support the potential applicability of MSC-CM-loaded PCL nanofibrous scaffold to treat peripheral nerve injury (PNI).