Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury. Display omitted •The bile canaliculi network displays geometric heterogeneity within the liver lobule•Bile flow is driven by osmotic effects and bile canaliculi contractility•A multi-scale model reveals gradients of bile velocity and pressure within the lobule•The multi-scale model predicts drug-induced alterations of bile flow Meyer et al. developed a 3D multi-scale model of biliary fluid dynamics in the mouse liver lobule to predict drug-induced alterations of bile flow. They demonstrate two inversely related gradients of bile velocity and pressure in the lobule and show that bile flow is driven by the osmotic effects of bile secretion and bile canaliculi contractility.