Hallmarks of neuronal differentiation are neurite sprouting, extension and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase 2 (CT2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth [Carter et al. (2003) J. Biol. Chem. 278:44988]. CT2 mRNA is highly expressed in the brain. We show that CT2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CT2 expression in PC12 cells differentiated by nerve growth factor. In CT2-silenced cells, numbers of primary and secondary neurites were markedly reduced suggesting that CT2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CT2-deficient cells. CT2 was not phosphorylated in vitro by Akt. However, inhibition of cdk5 by roscovitine blocked CT2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CT2 in neurons for promoting neurite outgrowth and branching, and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.