Background: Peripheral nervous system injuries are common and currently have no definitive treatment method. Phenytoin is one of the main antiepileptic drugs. Some studies have described a cerebroprotective effect of phenytoin in an established model of global cerebral ischemia. Objectives: In this study, the neuroprotective effects of phenytoin were evaluated on the cultivation and maintenance of Wharton’s jelly stem cells (WJSCs) on acellularized sciatic nerve scaffolds. Methods: In this study, acellular scaffolds from the rat sciatic nerve were prepared by the sondell method. After extraction of cells of MSCs, flow cytometry analysis was executed. Also, cell differentiation potential was evaluated by placement in osteogenic and adipogenic differentiation media for 21 days. Biocompatibility of the scaffold and cell viability were investigated using the MTT assay. The morphological and cell adhesion characteristics of MSCs on acellular scaffolds were compared using SEM micrographs images. Data were analyzed using the one-way analysis of variance (ANOVA) and Tukey post hoc test by SPSS (version 19.0) software. Results: The removal of cells from the scaffold was confirmed by stanning with hematoxylin-eosin, van Gieson's picro-fuchsin, and DAPI. With the aid of flow cytometry analysis and differentiation into bone and fat cells, it was confirmed that extracted cells were mesenchymal stem cells. The results of the MTT assay showed that phenytoin increased cell viability and retention on the scaffold. Conclusions: The study indicated that phenytoin improves the viability of cells and provided a good condition for the growth, survival, and attachment of cells to the scaffold when compared to the control group. These results suggest that phenytoin can be considered a new treatment for nerve regeneration and tissue engineering applications.