The loop diuretic furosemide has an action to inhibit Na+–K+–2Cl− co‐transporter at the thick ascending limb of Henle's loop resulting in diuresis. Furosemide also has the non‐diuretic effects by binding to GABA‐A receptor which may involve the gastrointestinal tract. The aim of this study was to investigate the effects of furosemide on smooth muscle contractions in mice ileum and proximal colon. Each intestinal segment suspended in an organ bath was connected to a force transducer. Signal output of mechanical activity was amplified and recorded for analysis using PowerLab System. After equilibration, the intestine was directly exposed to furosemide, GABA, GABA‐A receptor agonist (muscimol), or muscarinic receptor antagonist (atropine). Furosemide (50, 100 and 500 μmol L−1) acutely reduced the amplitude of ileal and colonic contraction. In the ileum, 1 mmol L−1 GABA and 10‐60 μmol L−1 muscimol significantly increased the amplitude, whereas in the colon, 50‐100 mmol L−1 GABA and 60 μmol L−1 muscimol decreased the contractions. The contractions were also significantly suppressed by atropine. To investigate the mechanisms underlying the inhibiting effect of furosemide, furosemide was added to the organ bath prior to the addition of muscimol or atropine. A comparison of furosemide combined with muscimol or atropine group and furosemide group showed no significant difference of the ileal contraction, but the amplitude of colonic contraction significantly decreased when compared to adding furosemide alone. These results suggest that furosemide can reduce the ileal and proximal colonic contraction mediated by blocking and supporting of GABA‐A receptor, respectively, resulting in decreased acetylcholine release.