Caspase-6 (CASP6) has an important role in axonal degeneration during neuronal apoptosis and in the neurodegenerative diseases Alzheimer and Huntington disease. Decreasing CASP6 activity may help to restore neuronal function in these and other diseases such as stroke and ischemia, where increased CASP6 activity has been implicated. The key to finding approaches to decrease CASP6 activity is a deeper understanding of the mechanisms regulating CASP6 activation. We show that CASP6 is posttranslationally palmitoylated by the palmitoyl acyltransferase HIP14 and that the palmitoylation of CASP6 inhibits its activation. Palmitoylation of CASP6 is decreased both in Hip14(-/-) mice, where HIP14 is absent, and in YAC128 mice, a model of Huntington disease, where HIP14 is dysfunctional and where CASP6 activity is increased. Molecular modeling suggests that palmitoylation of CASP6 may inhibit its activation via steric blockage of the substrate-binding groove and inhibition of CASP6 dimerization, both essential for CASP6 function. Our studies identify palmitoylation as a novel CASP6 modification and as a key regulator of CASP6 activity.Cell Death and Differentiation advance online publication, 2 December 2016; doi:10.1038/cdd.2016.139.