The tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr(−/−)) mice were crossbred with ATP binding cassette subfamily B member 4–deficient (Abcb4(−/−), alias Mdr2(−/−)) mice. Male and female wild-type, Tr(−/−), Mdr2(−/−), and Tr(−/−)Mdr2(−/−) mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2(−/−) and Tr(−/−)Mdr2(−/−) mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2(−/−) mice, compared with extensive bridging fibrosis in Tr(−/−)Mdr2(−/−) mice. DR cell population was greatly expanded in the Tr(−/−)Mdr2(−/−) versus Mdr2(−/−) mice. The expanded DR cell population in Tr(−/−)Mdr2(−/−) mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr(−/−)Mdr2(−/−) versus Mdr2(−/−) mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2(−/−) mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2(−/−) model of cholestasis.