Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr^(−/−) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr^(−/−) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr^(−/−) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr^(−/−) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr^(−/−) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr^(−/−) cells. Cftr^(−/−) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr^(−/−) cells compared with wild-type cells. Secreted mucins from Cftr^(−/−) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.