We tried to image obstructive cholestasis by using a newly developed imaging system to measure the alterations of hepatobiliary function in living mice with their bile ducts ligated. A hepatic imaging window was installed on the upper abdomen soon after the mice underwent ligation of common bile duct. On the next day, the mice received intravenous injection of rhodamine B isothiocyanate-dextran and carboxyfluorescein diacetate. The later would be transformed into fluorogenic carboxyfluorescein (detected at 500~550 nm) by hepatocytes and then excreted into bile canaliculi. The images were acquired by multiphoton microscopy. The fluorescence intensities at 500~550 nm within hepatocytes or sinusoids were measured in time series. In mice with bile duct ligation, bile canaliculi failed to appear during the whole observation period over 100 minutes post-carboxyfluorescein diacetate injection, while the fluorescence retained much longer within sinusoids. Furthermore, the fluorescence intensities in sinusoids were persistently higher than in hepatocytes during the course. Bile duct ligation impedes hepatocytes to excrete carboxyfluorescein into bile canaliculi. The kinetics of fluorescence intensities in hepatocytes and sinusoids indicated there is an active machinery operating backflow of this fluorogenic bile solute from hepatocytes into sinusoids in the liver with obstructive cholestasis.