Chronic hepatitis C virus (HCV) infection is characterized by progressive hepatic fibrosis, a process dependent on monocyte recruitment and accumulation into the liver. The mediators expressed in chronically injured liver that control the differentiation of human monocytes into profibrotic macrophages (Mφ) remain poorly defined. We report that chronically HCV-infected patients with high fibrosis stages have higher serum levels of macrophage colony-stimulating factor (M-CSF) and interleukin (IL)−34 than HCV-infected patients with lower fibrosis stages and healthy subjects. Immunohistochemistry reveals an intense expression of IL-34 and M-CSF by hepatocytes around liver lesions. In addition, HCV infection and inflammatory cytokines enhance the in vitro production of IL-34 and M-CSF by hepatocytes. We next analyzed the acquisition of profibrotic properties by Mφ generated with M-CSF (M-CSF-Mφ) or IL-34 (IL-34-Mφ). M-CSF and IL-34 up-regulate the expression, by differentiating monocytes, of chemokine (C-C motif) ligand (CCL)2, CCL4, C-C chemokine receptor (CCR)1, and CCR5, which are involved in monocyte recruitment/Mφ accumulation in liver lesions. M-CSF-Mφ and IL-34-Mφ also express the hepatic stellate cell (HSC) activators, platelet-derived growth factor, transforming growth factor beta, and galectin-3. IL-34-Mφ and M-CSF-Mφ induce type I collagen synthesis by HSCs, the main collagen-producing cells in liver fibrosis. IL-13, whose expression correlates with the fibrosis stage in HCV-infected patients, decreases the expression of the collagenase, matrix metalloproteinase 1, by IL-34-Mφ and M-CSF-Mφ, thereby enhancing collagen synthesis. By inhibiting the production of interferon-gamma (IFN-γ) by activated natural killer cells, IL-34-Mφ and M-CSF-Mφ prevent the IFN-γ-induced killing of HSCs. Conclusion: These results identify M-CSF and IL-34 as potent profibrotic factors in HCV liver fibrosis. (Hepatology 2014;60:1878–1889)