The functions of Cdk5/p35 have been uncovered through the identification of the substrates of Cdk5, most of which were first identified to interact with p35. The p35-associated Cdk5 activity is mainly localized to membrane fractions. Nonetheless, Cdk5 and p35 are also expressed in the nuclei of neurons. Thus, understanding the regulatory pathways that control the nucleocytoplasmic trafficking of Cdk5 and/or p35 may provide insights to their functional roles in neural development. It is noteworthy that the nuclear import of p35 is mediated by the importin pathway and that endogenous p35 is shuttled between the nucleus and cytoplasm upon growth factor stimulation. While the mechanism that regulates the nuclear export of p35 has not been investigated, the major regulatory mechanism of nuclear–cytoplasmic protein transportation is mediated by the nuclear export receptor, chromosome region maintenance 1 protein. CRM1 binds its cargo protein through recognition of a hydrophobic nuclear export signal peptide sequence. The present study aimed to discover novel roles of p35 in neural development through identification of its new interacting partner. We identified a new p35-interacting protein, nuclear hormone receptor coregulator -interacting factor 1. These findings reveal an unexpected role of p35 in mediating the nucleocytoplasmic shuttling of NIF-1. NIF-1 protein, which was originally identified to associate with NRC, is prominently expressed in the nuclear fractions of early differentiating neurons and involved in neurogenesis. The results show that p35 regulates the subcellular localization of NIF-1. Overexpression of p35 stimulates the nuclear export of NIF-1 via a CRM1-dependent pathway, and this nuclear/cytoplasmic shuttling of NIF-1 is mediated by the NES identified on p35. Importantly, inhibition of the CRM1-dependent pathway triggers the nuclear accumulation of p35 in neurons. These findings collectively reveal a previously unidentified role of p35 in the regulation of the nucleocytoplasmic trafficking of proteins and provide insights into how p35 regulates gene transcription. The neuronal activator of Cdk5, p35, is believed to exert its functions through its interaction with Cdk5, resulting in the phosphorylation of various cellular substrates. In the nucleus, Cdk5/p35 controls transcriptional regulation at multiple levels, including the phosphorylation of transcription factors and regulation of the histone acetylation through their interaction with histone deacetylase complexes. The present study identified a new mechanism of p35 wherein it regulates nuclear functions through modulating the nuclear accessibility of the transcriptional regulator NIF-1. In particular, p35 interacts with NIF-1 and regulates the subcellular localization of the protein independent of Cdk5-dependent phosphorylation. Thus, the present findings reveal a new mechanism of p35 in transcriptional regulation that does not require the kinase activity of Cdk5. Deficiency of p35 results in aberrant brain development and adult lethality, suggesting that the protein is critical for neural development. While p35 is ubiquitously expressed in neurons, its nuclear function is VE-822 evidenced by the functional importance of nuclear Cdk5/p35 in the maintenance of neuronal survival. Although the mechanism that regulates the subcellular distribution of p35 is poorly understood, post-translational modification of p35 by myristoylation is suggested to determine the attachment of the protein to the membrane. The present findings identified an NES on p35 that controls the nucleocytoplasmic shuttling of p35.