Updated project metadata. A cGMP signalling cascade composed of the ligand C-type natriuretic peptide (CNP), the natriuretic peptide receptor 2 (Npr2) and the cGMP-dependent kinase Iα (cGKI) is implicated by growth cone splitting in the bifurcation of afferents from dorsal root ganglia (DRG), cranial sensory ganglia (CSG) and mesencephalic trigeminal neurons (MTN). To get further mechanistic insights into the process of axon bifurcation we applied different cell culture approaches to decipher downstream activities of cGKI in somatosensory growth cones. We demonstrate that CNP induces an enlargement of DRG growth cones via cGKI which can be blocked or stimulated by pharmacological reagents that interfere with S-palmitoylation. cGKI colocalizes primarily in the central domain of the growth cone with the palmitoylome and vesicular structures including the endoplasmic reticulum, early endosomes, lysosomes and in the soma in the Golgi apparatus. Consistently, an acyl-biotin-exchange chemistry-based screen indicated that cGMP signalling regulates S-palmitoylation of a restricted pool of proteins in the DRG-derived cell line F11.