The effects of cyclophosphamide on 5-hydroxytryptamine (5-HT) synthesis in the intestinal tissue of rats were investigated. Rats received 120 mg/kg cyclophosphamide intraperitoneally as a single administration, and kaolin and food intake was measured by an automatic monitoring apparatus. Ileal tissues were collected at either 24 or 72 h after administration. Cyclophosphamide caused a significant increase in kaolin intake at the acute and the delayed phases and was associated with a decrease in food intake, and body weight. Cyclophosphamide had no significant effect on intestinal mucosal morphology, or inducible nitric oxide synthase and cyclooxygenase-2 expression in the intestine. Cyclophosphamide significantly increased tryptophan hydroxylase 1 (TPH1) mRNA expression, number of anti-TPH antibody-positive cells, and 5-HT content in the intestine. Cyclophosphamide also significantly increased the expression of Tac1 mRNA, encoding preprotachykinin-1, which is a preprotein of substance P, and the number of anti-substance P antibody-positive cells in the intestine. Cyclophosphamide significantly increased Lgr5, Bmi1, and Atoh1 mRNA levels, which are markers for the proliferation and differentiation of stem cells. This study demonstrated that cyclophosphamide induced pica in rats, and potentiated 5-HT synthesis associated with hyperplasia of substance P-containing enterochromaffin cells without causing severe intestinal injury.