Background Meibomian gland dysfunction (MGD) is the leading cause of evaporative dry eye and is characterized by gland obstruction, lipid imbalance, and tear film instability. While inflammatory mechanisms have been extensively studied, the molecular composition of meibum and its regulatory networks remain incompletely understood.Methods High-fidelity lipidomic profiling of meibum from MGD patients (n = 4) and healthy controls (n = 4) was performed using Q-TOF UPLC-MS with four-dimensional identification. Transcriptomic data from normal (n = 6) and MGD-affected (n = 6) meibomian glands were analyzed using GO, KEGG, and protein–protein interaction network analyses. Integrated lipid–enzyme–gene co-clustering was applied to construct regulatory networks.Results MGD samples showed enrichment of long-chain and neutral lipids, particularly cholesteryl esters, whereas controls displayed higher levels of short-chain ceramides and fatty acids. Dysregulated lipid species were enriched in structurally modified sphingolipids. Transcriptomic analysis revealed involvement of PI3K-Akt, MAPK, cytokine signaling, and extracellular matrix remodeling, together with altered transcriptional regulation and membrane transport.Conclusions Excess long-chain neutral lipids increase tear film viscosity and promote ductal obstruction, while short-chain and modified lipids support tear film stability. Coordinated disruption of lipid metabolism, cytoskeletal dynamics, and cellular homeostasis drives progressive gland dysfunction and chronic ocular surface damage in MGD.