INTRODUCTION: Improving fat absorption remains a challenge in cystic fibrosis (CF). Antibiotics (AB) treatment has been shown to improve body weight in CF mice. The mechanism may include improvement in fat absorption. We aimed to determine the effect of AB on fat absorption in two CF mouse models. RESULTS: AB did not improve total fat absorption. Interestingly, AB accelerated the absorption of isotope-labeled fats, in both Delta/Delta and WT mice. The changes observed were not related to the solubilization capacity of bile or to changes in the bacteria in the small intestine. AB reduced the fecal excretion of cholate by similar to 50% (P < 0.05) in both CF mouse models, indicating improved intestinal bile salt absorption. DISCUSSION: In conclusion, AB treatment does not improve total fat absorption in CF mice but does decrease fecal loss of bile salts and accelerate long-chain fatty acid (LCFA) absorption. METHODS: For 3 weeks, we administered oral AB (ciprofloxacin/metronidazole) or control treatment to homozygous Delta F508 (Delta/Delta), cystic fibrosis transmembrane conductance regulator (CFTR) knockout (-/-), and wild-type (WT) mice and quantified fat absorption using a 72-h fat balance test. In Delta/Delta mice, we assessed fat absorption kinetics by administering tri-1-C-13-palmitin and 1-C-13-stearate intragastrically and determining the appearance of stable isotope-labeled fats in plasma.