Symptoms of choledochal cysts are caused by protein plugs. We performed proteomic analysis of protein plugs to elucidate formation mechanism. Protein plugs were obtained from three pediatric patients with choledochal cyst. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel bands common to the samples were excised for mass spectrometry. Mass spectra were compared with the NCBI database for protein identification. Gel bands of protein plug samples were predominant at 14 kilodaltons (kDa), followed by 29 kDa. Four other thin bands were common to the plug samples. Four bands (including 14 and 29 kDa) were identified as lithostathine, and one band as serum albumin. Plugs consisted mostly of lithostathine, a protein secreted by pancreatic acinar cells into pancreatic juice. The mechanism involves trypsinogen and lithostathine regurgitating into the cyst through an aberrant union of pancreaticobiliary ducts. Activated trypsin cleaves soluble lithostathine into insoluble forms that aggregate to form plugs.