Background <p>Changes in macrophage function are crucial contributors to hepatic inflammation and fibrosis. However, the role of macrophages in the development of liver fibrosis in dairy cows with ketosis remains unclear. This study integrated proteomics and cytokine array approach to identify the multifactorial and multicellular interaction effects driving liver fibrosis in dairy cows with ketosis and analyze the mechanism by which the proinflammatory shift in macrophages contributes to liver fibrosis.</p> Results <p>Histopathological analysis revealed liver injury, including severe steatosis, infiltration of inflammatory cells, an increase in lipid deposition, and a decrease in glycogen expression in ketotic cows. Moreover, the number of mitochondria considerably increased in hepatocytes. The activation of the dynamin-related protein 1/mitochondrial fission factor (DRP1/MFF) pathway induced excessive mitochondrial fission, and the inhibition of the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) pathway led to the accumulation of intracellular reactive oxygen species (ROS). Proteomic analysis revealed the activation of extracellular matrix (ECM)-related functions and the NF-κB pathway in the liver, whereas cytokine array analysis revealed that the cytokine network was dysregulated. The accumulation of ROS triggered NF-κB nuclear translocation, inducing a proinflammatory shift in macrophages and liver inflammation. M1 polarization of macrophages promotes the release of proinflammatory mediators, which stimulated hepatic stellate cells (HSCs) activation, leading to ECM deposition, ultimately contributing to liver fibrosis.</p> Conclusions <p>To summarize, our study revealed the multifactorial and multicellular interaction effects driving liver fibrosis. Our results preliminarily showed that increased mitochondrial fission and inhibition of the Nrf2/HO-1 pathway are key factors in activating macrophages, which can lead to liver fibrosis in dairy cows with ketosis.</p>