The term blood-bile barrier (BBlB) refers to the physical structure within a hepatic lobule that compartmentalizes and hence segregates sinusoidal blood from canalicular bile. Thus, this barrier provides physiological protection in the liver, shielding the hepatocytes from bile toxicity and restricting the mixing of blood and bile. BBlB is primarily composed of tight junctions; however, adherens junction, desmosomes, gap junctions, and hepatocyte bile transporters also contribute to the barrier function of the BBlB. Recent findings also suggest that disruption of BBlB is associated with major hepatic diseases characterized by cholestasis and aberrations in BBlB thus may be a hallmark of many chronic liver diseases. Several molecular signaling pathways have now been shown to play a role in regulating the structure and function and eventually contribute to regulation of the BBlB function within the liver. In this review, we will discuss the structure and function of the BBlB, summarize the methods to assess the integrity and function of BBlB, discuss the role of BBlB in liver pathophysiology, and finally, discuss the mechanisms of BBlB regulation. Collectively, this review will demonstrate the significance of the BBlB in both liver homeostasis and hepatic dysfunction.