Lactylation is an emerging post-translational modification (PTM) that connects lactate metabolism with epigenetic regulation and protein-functional remodeling. Since histone lactylation was identified in 2019, accumulating evidence has shown that lactylation is not restricted to chromatin regulation, but also modifies non-histone proteins involved in protein stability, DNA repair, immune escape, metabolic adaptation, fibrosis, and tissue remodeling. However, current studies remain highly heterogeneous, and a central unresolved question is how the same lactate-derived modification generates distinct biological outcomes across cell types and disease contexts. In this review, we summarize the molecular machinery of lactylation, including writers, erasers, candidate readers, donor-generating routes, and histone and non-histone substrates, and propose a four-layer lactylation circuit as an integrative analytical framework. The framework comprises four regulatory layers: Layer I, metabolic input, which determines lactate production, transport, and compartmentalization; Layer II, donor substrate supply, which includes a putative lactyl-coenzyme A (Lac-CoA)-dependent route and an alanyl-tRNA synthetase 1 (AARS1)-dependent direct lactate-sensing route; Layer III, enzymatic and spatial control, which governs substrate selectivity through writers, erasers, and local molecular organization; and Layer IV, decoding and PTM crosstalk, which involves candidate reader proteins and competition or cooperation among lactylation, acetylation, ubiquitination, and other lysine modifications. We further discuss emerging mechanistic issues, including local Lac-CoA generation, Lac-CoA versus acetyl-coenzyme A competition, AARS1-mediated lactyl-adenylate formation, structural recognition of lactyl-lysine by BRG1, DPF2, TRIM33, and BRD9, and site-specific non-histone lactylation of proteins such as p53 and MRE11. These mechanisms suggest that lactylation output is determined not simply by bulk lactate abundance, but by the coordinated effects of donor availability, enzyme selectivity, subcellular localization, substrate identity, and downstream decoding. We then apply this framework to three major disease domains. In neurodegenerative diseases, lactylation links microglial glycolytic reprogramming with inflammatory and neurodegenerative outputs. In cancer, distinct lactylation circuits regulate proliferation, immune evasion, DNA repair, and metabolic reprogramming. In metabolic and cardiovascular diseases, chronic metabolic stress is translated into fibrosis, steatosis, endothelial-to-mesenchymal transition, vascular remodeling, calcification, or tissue repair. To provide complementary systems-level support, we analyzed publicly available transcriptomic datasets from gastric cancer, Alzheimer""s disease, and metabolic dysfunction-associated steatotic liver disease (MASLD). Differential-expression and pathway-enrichment analyses revealed partially overlapping metabolic, inflammatory, and tissue-remodeling signatures across these disease contexts. Nevertheless, we emphasize that bulk mRNA-level changes do not directly indicate protein lactylation abundance, site-specific modification stoichiometry, or causal lactylation mechanisms. Finally, we summarize therapeutic strategies targeting different circuit layers, including lactate dehydrogenase and monocarboxylate transporters at the metabolic-input level, ACSS2-dependent donor supply, p300/CBP-associated enzymatic control, and downstream PD-1/PD-L1 or G6PD/pentose phosphate pathway programs. These strategies should currently be regarded as lactylation-related rather than lactylation-specific interventions. The proposed four-layer circuit therefore serves as a hypothesis-generating framework rather than a fully validated universal mechanism. Future studies integrating isotope tracing, subcellular acyl-CoA metabolomics, site-resolved lactylomics, structural biology, reader-domain perturbation, and single-cell or spatial multi-omics in disease-relevant models will be essential to define causal circuit topology, identify context-specific therapeutic vulnerabilities, and determine whether lactylation can ultimately be translated into a clinically actionable regulatory axis.