In the context of cancer, miRNAs in serum from patients with breast cancer and diffuse large-B-cell lymphoma have been shown to be stable and highly predictive of malignancy and survival. These reports suggest significant roles for extracellular miRNAs, in addition to those of tissue miRNAs, in regulating many physiological processes. As this field develops, these miRNAs may become diagnostic and therapeutic targets in clinically relevant situations. These reports promoted us to speculate that miRNAs may be present and stable in human bile, just as they are in other bodily fluids, and that bile-borne miRNAs could be used as novel biomarkers for BTC. The major components of human bile are bile acid, cholesterol, bilirubin, bicarbonates, electrolytes, and water. Bile is produced by hepatocytes in the liver and flows via the bile duct into the duodenum, where it assists in lipid digestion and absorption. Like blood, bile can be collected from patients who undergo diagnostic and/or therapeutic bile drainage. One can therefore understand the utility of a BTC biomarker in bile and/or serum. A bile biomarker could expedite the diagnosis of BTC by prompting further histological examinations such as bile cytology, brush cytology, and forceps biopsy of the bile duct. In this study, we first examined whether miRNAs exist and can be detected in human bile by small RNA library sequencing. Next, we determined whether the expression of specific miRNAs in bile differs between patients with cancer and those without cancer using high-throughput real-time PCR-based miRNA expression microarrays. Finally, we assessed the potential of bile miRNAs as novel biomarkers for BTC. Here, we report on the presence and stability of bile miRNAs, their relative expression levels, as measured by high-throughput real-time PCR, and differences in the miRNA profiles between bile samples from patients with benign and malignant biliary tract disease. After confirming the existence of miRNAs in bile using specific miRNA primers, we performed a more comprehensive analysis using miRNA microarrays, which allowed us to test for the presence of 667 miRNA species. Although this is a small study, we have confidence that our concept of using miRNAs to distinguish benign/malignant biliary tract diseases will be validated as more large-scale studies are conducted in the future. In addition to proposing the diagnostic use of bile miRNAs, we have developed reliable methods for extracting and evaluating miRNAs that are compatible with clinical CHIR-99021 msds testing.