Objective To determine the genotypic and phenotypic characteristics of alkaline sphingomyelinase (alk-SMase) knockout (KO) mice and to provide the foundation data of this animal model for further study of the role of alk-SMase in colon cancer. Methods DNA was extracted from the tail tissue of wild type, heterozygous, homozygous KO mice, respectively. PCR analysis was used for genotyping. HE staining, confocal microscopy, and mass spectrometry were used for the detection of morphology, alk-SMase expression and sphingolipid metabolites in liver and intestine of the mice. Results Compared with the wild type and heterozygous mice, the homozygous KO mice showed that no changes occurred in the appearance and body weight, there was only one band (247bp) appeared on the genotyping, the thickness in small intestinal mucosa was significantly increased with a lower expression level of alk-SMase, and the amount of sphingolipid metabolites in the intestine and liver was changed, i.e. increase of SM and S1P, and reduction of ceramide. Conclusions Our findings demonstrate that the homozygous KO mice have specific genotype and phenotype that do not affect their growth. These mice will provide an ideal animal model for further study of alk-SMase functions.