Alkaline sphingomyelinase (Alk-SMase) is a key enzyme in the intestinal tract for digestion of dietary sphingomyelin (SM), which generates lipid messengers with cell-cycle regulating effects. The enzyme is significantly decreased in ulcerative colitis and colon cancer. Based on this information, we wanted to investigate whether the enzyme had preventive effects against murine colitis. We report herein a method to express a biologically active Alk-SMase from Pichia pastoris yeast cells. By using the expressed enzyme to treat a rat colitis model induced by dextran sulfate sodium, we found that intrarectal instillation of Alk-SMase once daily for 1 week significantly reduced the inflammation score and protected the colonic epithelium from inflammatory destruction. We found a tendency for decreased tumor necrosis factor (TNF)-alpha expression in the Alk-SMase-treated group. This study, for the first time, provides a method to produce the enzyme and shows the potential applicability of the enzyme in the treatment of inflammatory bowel diseases.