Protein lipidation, a prominent form of post-translational modification, involves the attachment of various lipids to proteins, enhancing their hydrophobicity and modulating their functions. This review comprehensively discusses the regulatory enzymes, catalytic mechanisms, and physiological and pathological implications of protein lipidation, focusing on S-palmitoylation. S-palmitoylation is catalyzed by DHHC palmitoyl S-acyltransferases (DHHC-PATs) and mediated by depalmitoylases. The process involves autopalmitoylation of DHHC-PATs followed by the transfer of palmitoyl chains to target proteins. DHHC-PATs are distributed across different organelles and exhibit substrate redundancy, complicating their individual characterization. Selective inhibitors of DHHC-PATs are still lacking, but broad-spectrum inhibitors like 2-bromopalmitate and cyanomethyl-N-myricrylamide (CMA) are available. Depalmitoylation is achieved by APTs, PPTs, and ABHD family thioesterases, each with specific substrate preferences. S-palmitoylation regulates protein trafficking, localisation, stability, conformation, and interactions, influencing cellular processes such as synaptic activity and immune responses. Abnormal S-palmitoylation has been linked to various diseases, including cancer and neurodegenerative disorders. In cancer, dysregulation of S-palmitoylation can promote tumour progression by enhancing the activity of oncogenic signalling pathways or protecting tumour suppressor proteins from degradation. In neurodegenerative diseases, abnormal S-palmitoylation contributes to protein aggregation and dysfunction. Targeting S-palmitoylation represents a promising therapeutic strategy, with several agents, such as asciminib and lonafarnib, already approved for clinical use.Protein lipidation, a prominent form of post-translational modification, involves the attachment of various lipids to proteins, enhancing their hydrophobicity and modulating their functions. This review comprehensively discusses the regulatory enzymes, catalytic mechanisms, and physiological and pathological implications of protein lipidation, focusing on S-palmitoylation. S-palmitoylation is catalyzed by DHHC palmitoyl S-acyltransferases (DHHC-PATs) and mediated by depalmitoylases. The process involves autopalmitoylation of DHHC-PATs followed by the transfer of palmitoyl chains to target proteins. DHHC-PATs are distributed across different organelles and exhibit substrate redundancy, complicating their individual characterization. Selective inhibitors of DHHC-PATs are still lacking, but broad-spectrum inhibitors like 2-bromopalmitate and cyanomethyl-N-myricrylamide (CMA) are available. Depalmitoylation is achieved by APTs, PPTs, and ABHD family thioesterases, each with specific substrate preferences. S-palmitoylation regulates protein trafficking, localisation, stability, conformation, and interactions, influencing cellular processes such as synaptic activity and immune responses. Abnormal S-palmitoylation has been linked to various diseases, including cancer and neurodegenerative disorders. In cancer, dysregulation of S-palmitoylation can promote tumour progression by enhancing the activity of oncogenic signalling pathways or protecting tumour suppressor proteins from degradation. In neurodegenerative diseases, abnormal S-palmitoylation contributes to protein aggregation and dysfunction. Targeting S-palmitoylation represents a promising therapeutic strategy, with several agents, such as asciminib and lonafarnib, already approved for clinical use.