Dogs represent a promising animal model for analyzing human neurodegenerative diseases, owing to their similarities to humans in nervous system architecture and behavioral phenotypes. Neural stem cells (NSCs) serve as a highly valuable in vitro experimental model for investigating neurogenesis, neurodegenerative disease pathogenesis, and neural molecular biology; however, studies on immortalized canine neural stem cell lines remain scarce. Herein, we successfully established an immortalized canine hippocampal neural stem cell line that can be continuously passaged in vitro via SV40 large T antigen (SV40LT) viral infection and subsequent cellular transformation. Both the immortalized NSCs and their normal parental counterparts differentiated into neuronal and glial lineages under induced differentiation conditions. Normal canine hippocampal NSCs can be passaged for no more than 10 generations, whereas the immortalized line can be passaged indefinitely while maintaining a normal karyotype. This immortalized canine hippocampal NSC line can act as a critical experimental tool for future research into neural differentiation mechanisms and stem cell-derived therapeutic strategies for neurological disorders in dogs.