Mixed lineage kinase domain-like (MLKL) is a key signaling protein of necroptosis. Upon activation by phosphorylation, MLKL translocates to the plasma membrane and induces membrane permeabilization which contributes to the necroptosis-associated inflammation. Membrane binding of MLKL is initially initiated by the electrostatic interactions between the protein and membrane phospholipids. We previously showed that MLKL and its phosphorylated form (pMLKL) are S-acylated during necroptosis. Here, we characterize acylation sites of MLKL and identify multiple cysteines that can undergo acylation with an interesting promiscuity at play. Our results show that MLKL and pMLKL undergo acylation at a single cysteine, C184, C269 and C286 are the possible acylation sites. Using all atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane level. Through systematic investigations of the S-palmitoyltransferases that might acylate MLKL in necroptosis, we showed that zDHHC21 activity has the strongest effect on pMLKL acylation, inactivation of which profoundly reduced the pMLKL levels in cells and improved membrane integrity. These results suggest that blocking the acylation of pMLKL destabilizes the protein at the membrane interface and causes its degradation, ameliorating necroptotic activity. At a broader level, our findings shed light on the effect of S-acylation on MLKL functioning in necroptosis and MLKL-membrane interactions mediated by its acylation.