We previously showed that dietary sphingomyelin (SM) inhibited cholesterol absorption in animals. The key enzyme hydrolyzing SM in the gut is alkaline sphingomyelinase (alk-SMase, NPP7). Here using fecal dual-isotope ratio method we compared cholesterol absorption in the wild type (WT) and alk-SMase knockout (KO) mice. The animals were fed an emulsion containing ¹⁴C-cholesterol and ³H-sitosterol. The radioactivities in the lipids of the fecal samples collected 4, 8 and 24 h thereafter were determined and the ratio of ¹⁴C/³H calculated. We found that the fecal ¹⁴C-cholesterol recovery in the KO mice was significantly higher than in the WT mice. Maximal 92% increase occurred 8 h after feeding. Recovery of ³H-sitosterol did not differ between the two groups. Accordingly the ¹⁴C/³H ratio of fecal lipids was 133% higher at 8 h and 75% higher at 24 h in the KO than in the WT mice. Decreased ¹⁴C-cholesterol was also found in the serum of the KO mice 4 h after feeding. Supplement of SM in the emulsion reduced the differences in fecal ¹⁴C-cholesterol recovery between the WT and KO mice due to a greater increase of ¹⁴C-cholesterol recovery in the WT mice. Without treatment the KO mice had significantly higher SM levels in the intestinal content and feces, but not in the intestinal mucosa or serum. The expression of NPC1L1 in the small intestine was not changed. In conclusion, alk-SMase is a physiological factor promoting cholesterol absorption by reducing SM levels in the intestinal lumen.