Abstract: Liver zonation refers to the spatial organization principle underlying core hepatic functions, specifically characterized by the gradient distribution of gene expression and metabolic activities in hepatocytes along the portocentral axis. This phenomenon is far from a static anatomical division; rather, it constitutes a dynamically and precisely regulated microenvironmental adaptive system. Research has revealed that the establishment and maintenance of zonation depend on finely tuned gradients of signaling pathways, such as Wnt/β-catenin, and regulatory networks formed by non-parenchymal cells within the microenvironment. This spatial optimization allocates key functions—including glucose and lipid metabolism, detoxification, and biosynthesis—thereby forming the fundamental architectural basis for the liver’s diverse and complex biochemical activities. In recent years, single-cell and spatial multi-omics technologies have unveiled the molecular blueprint of zonation with unprecedented resolution. Significantly, studies have identified zonation disruption as a critical pathological feature in the development and progression of various liver diseases, such as metabolic dysfunction-associated steatotic liver disease (MASLD), liver fibrosis, and hepatocellular carcinoma. Consequently, indepth analysis of liver zonation not only elucidates hepatic physiology but also provides a novel paradigm for understanding disease mechanisms. Targeting the zonation regulatory network to restore the spatial functional order of hepatocytes has emerged as a promising new strategy for precision therapy. This review aims to systematically elucidate the anatomical and molecular foundations of liver zonation, its metabolic characteristics, and its remodeling in disease states, thereby providing a theoretical framework for the future development of novel diagnostic and therapeutic strategies based on zonation biology.