Mucin, a major component of mucus, plays an important role in gallstone formation. The molecular mechanisms of mucin overproduction, however, still remain unknown. Several mucin genes (MUC) have been implicated in various diseases and gel-forming mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) were recognized to be the important components of digestive mucus. Furthermore epidermal growth factor receptor (EGFR) might regulate the function of MUC5AC. The aim of this study was to investigate the expression of MUC5AC mRNA and the possible role of EGFR in the function of MUC5AC. Total twelve patients underwent elective laparoscopic cholecystectomy due to gallstone disease were enrolled (age: 64.6 ± 15.5 years). The control group included two patients who underwent cholecystectomy without stone. The expression levels of MUC5AC and EGFR mRNAs were analyzed in gallbladder tissues using real-time reverse transcription-polymerase chain reaction assay. The expression levels of MUC5AC mRNA were increased in gallstone patients compared with those in control subjects, ranging from 2.5 to 1558 folds (mean 512.8 ± 493.6 folds, p = 0.004). In contrast, the expression levels of EGFR mRNA were decreased in all patients (mean 0.378 ± 0.322 fold, p = 0.002), and negative correlation was found between MUC5AC and EGFR (p = 0.02). There was no significant difference according to age, body mass index, and stone composition in the expression levels of MUC5AC and EGFR mRNAs. In conclusion, MUC5AC is over-expressed in gallstone disease, despite the decrease in the expression of EGFR mRNA. MUC5AC may be related to mucus hypersecretion.