Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decreases biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 h and up to 14 days. MC infiltration was evaluated by toluidine blue. BDL rats were perfused using standard collagenase perfusion. Following enzymatic digestion, tissue was passed through a fine gauge needle. Suspensions were incubated with MAb AA4, washed and incubated with goat anti-mouse-coated Dynal beads. MCs were stained with toluidine blue, and in isolated MCs the expression of FCɛRI and MC proteases was measured. The expression of histidine decarboxylase, histamine receptors, VEGF receptors, and TIE 1 and 2 was evaluated by qPCR. Histamine and VEGF-A secretion was measured in MC supernatants. MC purity was evaluated by CK-19, CK-8, albumin, VAP-1, and α-SMA expression. In vitro, cholangiocytes and HSCs were treated with isolated MC supernatants from BDL rats treated with either NaCl or cromolyn sodium (to block MC histamine release) and biliary proliferation and hepatic fibrosis were measured. MCs infiltrate the liver and surround bile ducts starting at day 2. We isolated a virtually pure preparation of mature, functional MCs. TEM images reveal distinct secretory granules and isolated MCs secrete histamine. MCs express FCɛRI, chymase, tryptase, RMCP-I, and RMCP-II, but were virtually void of other cell markers. Biliary proliferation and fibrosis increased following treatment with MC supernatants from BDL rats+NaCl and these parameters decreased in cells treated with MC supernatants from BDL+cromolyn sodium. In conclusion, we have isolated and characterized MCs from cholestatic livers. MCs regulate cholestatic liver injury and hepatic fibrosis. This tool provides a better understanding of the paracrine influence of mast cells on biliary/liver pathologies.