Lipid metabolites are emerging as pivotal regulators of protein function and cell signaling. The availability of intracellular fatty acid is tightly regulated by glycolipid metabolism and may affect human body through many biological mechanisms. Recent studies have demonstrated palmitate, either from exogenous fatty acid uptake or fatty acid synthesis, may serve as the substrate for protein palmitoylation and regulate protein function via palmitoylation. Palmitoylation, the most-studied protein lipidation, encompasses the reversible covalent attachment of palmitate moieties to protein cysteine residues. It controls various cellular physiological processes and alters protein stability, conformation, localization, membrane association and interaction with other effectors. Dysregulation of palmitoylation has been implicated in a plethora of diseases, such as metabolic syndrome, cancers, neurological disorders and infections. Accordingly, it could be one of the molecular mechanisms underlying the impact of palmitate metabolite on cellular homeostasis and human diseases. Herein, we explore the relationship between lipid metabolites and the regulation of protein function through palmitoylation. We review the current progress made on the putative role of palmitate in altering the palmitoylation of key proteins and thus contributing to the pathogenesis of various diseases, among which we focus on metabolic disorders, cancers, inflammation and infections, neurodegenerative diseases. We also highlight the opportunities and new therapeutics to target palmitoylation in disease development.