Niemann-Pick type C (NPC) disease is a fatal autosomal recessive neurodegenerative disorder caused, most commonly, by mutations in the NPC1 gene. At the cellular level, the disease is characterized by the storage of multiple lipids in the endosomal-lysosomal system, including free cholesterol, glycosphingolipids, sphingomyelin and the catabolic product of sphingolipids, sphingosine. Therapeutic options for NPC disease are relatively limited. One drawback for the development of novel therapies is the lack of suitable human neuronal cell models. In this work, a stable SH-SY5Y cell model for NPC disease was generated using short hairpin RNAs. An inhibition of the NPC1 expression of around 90% was obtained at the RNA level. The NPC1 knockdown was confirmed at the protein level. To characterize the stable cell line generated, cholesterol levels were analyzed in the NPC1-knockdown SH-SY5Y cells by filipin staining and gas chromatography-mass spectrometry. A characteristic NPC pattern and a twofold increase of the free cholesterol levels, related to intact SH-SY5Y cells, were found. Moreover, sphingolipids were analyzed by liquid chromatography-mass spectrometry and an increase in ganglioside GM2 levels was observed. The stable NPC1-knockdown SH-SY5Y cell line generated in the present study provides a human neuronal cell model for this lethal disease that could be a valuable tool for the study of future therapeutic approaches.