Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase beta2 (CTbeta2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988-44994). CTbeta2 mRNA is highly expressed in the brain. We show that CTbeta2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTbeta2 expression in PC12 cells differentiated by nerve growth factor. In CTbeta2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTbeta2 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 CTbeta2-deficient cells. CTbeta2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTbeta2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTbeta2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.