Previously, we reported that a null mutation of the Zdhhc13 gene caused by ethylnitrosourea (ENU) mutagenesis in mice resulted in severe systematic phenotypes with amyloidosis, alopecia, dermatitis and osteoporosis. In this study we continued to delineate the pathological mechanism for the dermatitis phenotype and to explore potential palmitoylation substrates of ZDHHC13, which potentially explain the loss-of-function phenotype of ZDHHC13 in skin. Our data clearly suggested that protein S-palmitoylation of a constellation of skin barrier components by ZDHHC13 is crucial for their protein stability, functions, and overall barrier integrity.