Gram-negative bacterial infections can cause varying degrees of liver injury in chickens. Although andrographolide has been shown to have a protective effect on the liver, its underlying mechanism of action and effects on liver proteins are not known. The study objectives were to analyze the actions of andrographolide on lipopolysaccharide (LPS)-induced chicken leghorn male hepatoma (LMH) cell injury, identify the different proteins in different groups using TMT proteomics, and explore the pharmacological effects and potential targets of andrographolide in LPS-induced liver injury. It was found that andrographolide reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the cell supernatant and alleviated LPS-induced injury in LMH cells. Proteomic analysis identified 50 and 166 differentially expressed proteins in the LPS vs NC group and LPS-Andro vs LPS group, respectively. Andrographolide may be involved in steroid metabolic processes, negative regulation of MAPK cascade, oxidative stress, and other processes to protect against LPS-induced liver injury. HMGCS1, HMGCR, FDPS, PBK, CAV1, PRDX1, PRDX4, and PRDX6, which were identified by differential proteomics, may be the targets of andrographolide. Our study may provide new theoretical support for andrographolide protection against liver injury.