Nerve growth factor (NGF) promotes neurite outgrowth through regulating cytoskeletal organization and cell adhesion. These activities are modulated by protein phosphorylation. Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-like kinase that phosphorylates several transcription factors. Although NLK is known to be expressed at relatively high levels in the nervous system, its function is not well understood. We found that NGF promotes the translocation of NLK to PC12 cells' leading edges, and triggers NLK kinase activity in them. Activated NLK directly phosphorylates microtubule-associated protein-1B (MAP1B) and the focal adhesion adaptor protein, paxillin. Knockdown of NLK attenuates the phosphorylation of both paxillin and MAP1B and inhibits both the NGF-induced re-distribution of F-actin and neurite outgrowth. We also discovered that NLK is a LiCl-sensitive kinase. LiCl is known to block NGF-induced neurite outgrowth and the phosphorylation of MAP1B and paxillin in PC12 cells. Therefore, the effects of LiCl are mediated in part by blocking NLK activity. These results suggest that NLK controls the dynamics of the cytoskeleton downstream of NGF signaling. © 2009 International Society for Neurochemistry.