Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase (PAT), ZDHHC21, in mediating signaling events required for gut hyperpermeability induced by inflammation. Using qPCR, we show that ZDHHC21 mRNA, production was enhanced by two-fold when intestinal epithelial cells were treated with TNFα/IFN in vitro. In addition, pharmacological targeting of PATs with 2-bromopalmitate (2-BP) showed significant improvement in TNFα/IFN mediated epithelial barrier dysfunction by using electric cell-substrate impedance sensing (ECIS) assays, as well as FITC-dextran permeability assays. Using the ABE assay and click chemistry, we show that TNFα/IFN treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins, and this response is inhibited by 2-BP. Using ZDHHC21 deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, H&E staining of small intestine, as well as transmission electron microscopy (TEM), showed mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury.