With the increasing use of bone marrow mesenchymal stem cells (BMSCs) in cell therapies, factors regulating BMSC differentiation have become the interest of current research. In this study, we investigated the effects of glial cell-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) on the course of BMSC differentiation. BMSCs were isolated from rat bone marrow and transfected with GDNF and NT-3 genes. Compared to mock-transfected BMSCs, GDNF and NT-3 induced BMSC differentiation to reveal neuron-like characteristics, i.e., the positive expression of neuronal marker MAP-2 and astrocyte marker GFAP, as detected by immunofluorescence assays. Semi-quantitative polymerase chain reaction (PCR) and western blot analyses showed that the increase of expression of GDNF and NT-3 in BMSCs also simultaneously elevated the mRNA expression of NSE, nestin, and MAP-2. Furthermore, the cell patch-clamp test demonstrated that the overexpression of GDNF and NT-3 in BMSCs enhanced voltage-activated potassium currents, implying that BMSCs possess great potential as a cell-based therapeutic candidate to treat neurological diseases.