This study was designed to test the in vivo efficacy of the chemical chaperone trimethylamine oxide (TMAO) in correcting the Cl⁻ transport defect in a mouse model of cystic fibrosis (CF). Rectal potential difference (RPD) measurements were done in matched wild-type and ΔF508 CF mice. Mice were treated by subcutaneous injections of TMAO. Wild-type mice demonstrated a forskolin-stimulated, Cl⁻-dependent hyperpolarization of −6.4 ± 0.8 mV (n = 11), which was significantly increased to −13.1 ± 1.4 mV after treatment with TMAO. ΔF508 CF mice showed no significant responses to forskolin. Treatment with TMAO recovered a forskolin-activated RPD in ΔF508 CF mice (−1.1 ± 0.2 mV; n = 17) but not in CFTR null mice. The effects of TMAO were dose dependent, resulting in a slope of −0.4 ± 0.1 mV · g⁻¹ · kg⁻¹ in ΔF508 CF mice. The forskolin-stimulated RPD in TMAO-treated ΔF508 CF mice was partially blocked by glibenclamide and further stimulated by apigenin. The total response to forskolin plus apigenin was −2.5 ± 0.45 mV (n = 6 mice), corresponding to 39% of the response evoked by forskolin only in wild-type mice.