Cholangiocarcinoma is an aggressive tumor with a tendency for local invasion and distant metastases. However, at present, there is no available tumor marker that can differentiate cholangiocarcinoma from benign bile duct disease. This study was designed to determine whether the serum levels of MMP7 can discriminate cholangiocarcinoma patients from benign biliary tract disease patients in comparison to carbohydrate antigen 19- 9 (CA19-9). This study was divided into 2 parts. Part 1 (a retrospective nonconsecutive case-control study), we measured the level of CA19-9 and MMP7 in the serum derived from the serum bank (44 cholangiocarcinoma and 36 benign biliary tract diseases patients). Part 2 (prospective consecutive cases from patients with symptoms of biliary tract disease), a total of 187 obstructive jaundice patients was consecutively enrolled and their values of serum MMP7 was assayed and compared with serum CA19-9. For part 1 study, a receiver operating characteristic (ROC) curve analysis revealed that the detection of the serum MMP7 level is reasonably accurate in differentiating cholangiocarcinoma from benign biliary tract disease patients (area under curve=0.73; 95% CI = 0.614-0.848). While the areas under the curve of the ROC curves for CA19-9 were 0.63 (95% CI = 0.491-0.761). For part 2 study, we found that MMP7 and CA19-9 serum levels were significantly elevated in cholangiocarcinoma patients (p<0.001). The area under the curve (AUC) from a receiver operating characteristic (ROC) curve analysis for the diagnosis of cholangiocarcinoma, using MMP7 was more accurate than CA19-9 (AUC =0.84; CI 95% 0.778-0.903 for MMP7 and AUC = 0.79; CI 95% 0.708-0.868 for CA19-9). The sensitivity and specificity of serum MMP7 (cut-off value of 5.5 ng/ml) was 75% and 78%, respectively, while the sensitivity and specificity of serum CA19-9 (cut-off value of 100 U/ml) was 68% and 87%, respectively. We concluded that serum values of MMP7 and CA19-9 appear to be useful biomarkers for differentiating cholangiocarcinoma from benign biliary tract diseases.