Cyclin-dependent kinase 5 (Cdk5), a neuronal Cdc2-like kinase, exhibits a variety of functions in neuronal differentiation and neurocytoskeleton dynamics, as well as neuronal degeneration. However, its role and induction mechanisms in retinoic acid (RA)-induced neuronal differentiation have not been well understood. In this study we newly found that RA treatment of SK-N-BE(2)C, human neuroblastoma cells, increased the expression of Cdk5 and its neuron specific activator p35 through the extracellular-signal-regulated kinase1/2 (ERK1/2) and cAMP-dependent protein kinase A (PKA) pathway. Inhibition of Cdk5 activity either by an inhibitor, roscovitine, or by transfection with a dominant negative form of Cdk5 caused a dramatic decrease in RA-induced differentiation, suggesting the requirement of Cdk5 kinase activity for the RA-induced neurite outgrowth. Furthermore, Cdk5 and p35 expression was decreased by ERK1/2 inhibition with PD98059 and increased by overexpression of a constitutive active mitogen-activated protein kinase kinase 1 (MEK1) mutant, suggesting the critical role of ERK1/2 in the induction of Cdk5 and p35. In addition, a transcription factor early growth response 1 (Egr-1) was induced by RA through the ERK1/2 pathway, suggesting its possible involvement in the p35 induction. RA treatment also induced c-fos mediated AP-1 binding, and cAMP-responsive element binding protein (CREB) mediated CRE binding via ERK1/2 and PKA pathway, respectively, in the Cdk5 promoter region, resulting in the induction of Cdk5. Our results suggest that ERK1/2 and PKA-induced regulation of Cdk5 activity possibly through Egr-1, c-fos, and CREB plays a critical role in the RA-induced neuronal differentiation.