Small intestinal bacterial overgrowth (SIBO) may play an important role in the gastrointestinal complications of cystic fibrosis (CF). This work explored two potential factors in development of SIBO in the CF (cftr^tm1UNC) mouse: impaired Paneth cell innate defenses and altered gastrointestinal motility. Postnatal differentiation of Paneth cells was followed by Defcr, Lyzs, and Ang4 gene expression, and SIBO was measured by quantitative PCR of the bacterial 16S rRNA gene. Paneth cell gene expression was low in 4 day old CF and wild type (WT) mice and increased similarly in both groups of mice between 12-16 days. Peak Paneth cell gene expression was reached by 40 days of age and was less for Defcr and Lyzs in CF mice compared to WT, where as Ang4 levels were greater in CF mice. SIBO occurred by postnatal day 8 in CF mice which is before Paneth cell development. Using gavaged rhodamine-dextran to follow motility, gastric emptying in CF mice was slightly decreased compared to WT, and small intestinal transit was dramatically less. Since antibiotics improve weight gain in CF mice, their effects on gastric emptying and small intestinal transit were determined. Antibiotics did not affect gastric emptying or transit in CF mice, but did significantly slow intestinal transit in WT mice suggesting a potential role of normal microflora in regulating transit. In conclusion, small intestinal transit was significantly slower in CF mice and this is likely a major factor in SIBO in CF.