Background/Objectives: Elevated concentrations of Hepatocarcinoma–Intestine –Pancreas/Pancreatitis-associated Protein I (HIP/PAP-I) in pancreatic juice have been reported in patients with pancreatic adenocarcinoma and have been considered as a promising tumor marker. This study was conducted to investigate whether biliary HIP/PAP-I can be used in the differential diagnosis of the cause of biliary obstruction. Methods: Bile was obtained from patients with bile duct obstruction on the day of biliary drainage. The etiology of biliary obstruction included gallstones ( n = 131), pancreatic cancer ( n = 32), cholangiocarcinoma ( n = 47), papilla Vater cancer ( n = 13), hepatocellular carcinoma ( n = 4) and metastatic cancer ( n = 16). In addition to HIP/PAP-I, the samples were analyzed for amylase to check for the presence of pancreaticobiliary reflux. Results: The biliary concentration of HIP/PAP-I was not statistically different between patients with gallstones (median, 9.70 ng/mL; interquartile range [IQR] 1.80–45.75) and cancers (median, 12.70 ng/mL; IQR, 3.85–36.75), P > 0.05. However, the amylase activity in the bile was markedly elevated in patients with gallstones (median, 228 U/L; IQR, 40–1965), compared to those with cancer (median, 32 U/L; IQR; 30–176), P < 0.001. The area under the ROC curve of amylase was 0.751 (95% CI: 0.69 to 0.81). At a cut-off value of 46 U/L, the biliary amylase distinguished patients with malignant obstruction from those with benign obstruction with a sensitivity of 66% and a specificity of 74%. Conclusions: Our data suggest that the biliary HIP/PAP-I measurement is not useful for differentiating causes of biliary obstruction. The divergent extent and duration of biliary obstruction caused by neoplasm and gallstones may contribute to the significant difference in the amylase activity in bile. Thus, amylase in bile represents a candidate marker in the differential diagnosis of the cause of biliary obstruction.