Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and post translational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSPI domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth. (C) 2017 Elsevier Inc. All rights reserved.