Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral sphingomyelinase-2 (nSMase2), an enzyme generating ceramide from sphingomyelin (SM), as a potential repressor for heptocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5 (CerS5). fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplet, suggesting nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs), and higher levels of phosphorylated signal transducer and activator of transcription 3 (pStat3), an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis.