It has been shown that Ouabain (OUA), a cardiotonic steroid, can bind to Na,K-ATPase (NKA) and trigger many signaling pathways in adult rat hippocampus such as cAMP response element-binding protein (CREB), Wnt/β-catenin and NF-κB in neurons, increasing Brain-derived neurotrophic factor (BDNF) release, neuronal dendritic branching and leading to a spatial long-term memory improvement. This study aimed to verify whether OUA affects neural precursor cells in the dentate gyrus, which is the main site of adult hippocampal neurogenesis. Primary cultures of neural precursor cells (NPCs) were made from newborn Wistar rat dentate gyri. These results suggest that OUA increases Neurod2 transcription in NPCs, but persistently decreases the release of NT3. Furthermore, OUA increased BDNF and nerve growth factor (NGF) levels and the percentage of generated neurons. These neurons also had a more developed dendritic arbor than the control group. In conclusion, 10 nM OUA increased NPCs differentiation into neurons, suggesting a new role for NKA in neurogenesis.