Purpose: Spinal cord injury (SCI) is damage to the spinal cord that resulted in irreversible neuronal loss, glial scar formation and axonal injury. Herein, we used the human amniotic fluid mesenchymal stem cells (hAF-MSCs) and their conditioned medium (CM), to investigate their ability in neuroblast and astrocyte production as well as functional recovery following SCI. Methods: Fifty-four adult rats were randomly divided into nine groups (n=6), included: Control, SCI, (SCI + DMEM), (SCI + CM), (SCI + MSCs), (SCI + Astrocyte), (SCI + Astrocyte + DMEM), (SCI + Astrocyte + CM) and (SCI + Astrocyte + MSCs). Following laminectomy and SCI induction, DMEM, CM, MSCs, and astrocytes were injected. Western blot was performed to explore the levels of the Sox2 protein in the MSCs-CM. The immunofluorescence staining against doublecortin (DCX) and glial fibrillary acidic protein (GFAP) was done. Finally, Basso-Beattie-Brenham (BBB) locomotor test was conducted to assess the neurological outcomes. Results: Our results showed that the MSCs increased the number of endogenous DCX-positive cells and decreased the number of GFAP-positive cells by mediating juxtacrine and paracrine mechanisms (P<0.001). Transplanted human astrocytes were converted to neuroblasts rather than astrocytes under influence of MSCs and CM in the SCI. Moreover, functional recovery indexes were promoted in those groups that received MSCs and CM. Conclusion: Taken together, our data indicate the MSCs via juxtacrine and paracrine pathways could direct the spinal cord endogenous neural stem cells (NSCs) to the neuroblasts lineage which indicates the capability of the MSCs in the increasing of the number of DCX-positive cells and astrocytes decline.Keywords: Astrocyte; Conditioned medium; Mesenchymal stem cells; Neuroblast; Spinal cord injury.