5‐fluorouracil (5‐FU) is part of the standard care for cancer treatment but is associated with high incidences of appetite loss and reduced food intake, which may contribute to chemotherapy‐induced cachexia (weakness and wasting of tissue). The role of gastrointestinal satiety hormones in chemotherapy‐induced appetite loss has not been intensively investigated. Peptide YY (PYY) and glucagon‐like peptide (GLP)‐1 are important signals of gastrointestinal satiety, so this study examined the roles of these gut hormones in 5‐FU‐induced reduction of dietary intake. Mice were given 5‐FU (50 mg/kg, intraperitoneal [i.p.]) every day for 4 consecutive days. Gene expression levels of proglucagon (Pro‐Gcg), a precursor of GLP‐1, and PYY in the colon were examined by real‐time RT‐PCR. Serum levels of GLP‐1 and PYY were measured by enzyme‐linked immunosorbent assay. Some mice were pretreated with the GLP‐1 receptor antagonist exendin9–39 (1 mg/kg) or the neuropeptide Y type 2 (NPY2) receptor antagonist BIIE0246 (2 mg/kg) via the i.p. route 30 minutes before 5‐FU administration. Mice receiving 5‐FU exhibited a significant reduction in food intake that was correlated with body weight loss. These mice also showed significantly enhanced expression levels of mRNAs encoding pro‐GLP‐1 and PYY in the transverse and distal colon as well as elevated serum concentrations of GLP‐1 and PYY compared to vehicle‐treated controls. The 5‐FU‐induced reduction in food intake was attenuated by BIIE0246 but not by exendin9–39. These data suggest that administration of a NPY2 receptor antagonist may be effective for attenuating the anorexia caused by 5‐FU chemotherapy.