Palmitoylation is required for the activities of several cancer-associated proteins, making the palmitoyl acyltransferase (PAT) enzymes that catalyze these reactions potential targets for anticancer therapeutics. In this study, we sought to identify and characterize a human PAT with activity toward N-terminally myristoylated and palmitoylated proteins. NIH/3t3 cells were stably transfected with vectors containing no insert, wild type human DHHC20, or a serine-substituted DHHS20 mutant. Compared with control cells, cells overexpressing wild-type DHHC20 displayed an increase in palmitoylation activity toward a peptide that mimics the N-terminus of myristoylated and palmitoylated proteins, but had no change in activity toward a peptide that mimics the C-terminus of farnesylated and palmitoylated proteins. Cells expressing DHHS20 had no significant change in activity toward either peptide. Overexpression of DHHC20 also caused phenotypic changes consistent with cellular transformation, including colony formation in soft agar, decreased contact inhibition of growth, and increased proliferation under low-serum conditions. Quantitative polymerase chain reaction analyses of human tissues demonstrated that DHHC20 is expressed in a tissue-specific manner, and is overexpressed in several types of human tumors, including ovarian, breast and prostate. Overall, these results demonstrate that DHHC20 is a human N-terminal-myristoyl-directed PAT involved in cellular transformation, that may play a role in cancer.