AbstractRegulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (DSCR) on human chromosome 21 and is related to the Down syndrome (DS) phenotype. To identify a novel binding partner of RCAN1, we performed yeast two‐hybrid screening and identified mitogen‐activated protein (MAP) kinase/extracellular signal‐regulated kinase (ERK) kinase 1 (MEK1) as a partner. MEK1 was able to bind and phosphorylate RCAN1 in vitro and in vivo. MEK1‐dependent RCAN1 phosphorylation caused an increase in RCAN1 expression by increasing the protein half‐life. Nerve growth factor (NGF)‐dependent activation of the MEK1 pathway consistently induced RCAN1 expression. Moreover, we found that RCAN1 overexpression inhibited NGF‐induced neurite outgrowth and expression of neuronal marker genes, such as growth cone‐associated protein 43 (GAP43) and synapsin I, via inhibition of MEK1‐ERK1/2 pathways. Our findings provide evidence that MEK1‐dependent RCAN1 phosphorylation acts as an important molecular mechanism in the control of neuronal differentiation.