The neuronal nuclear matrix protein, NRP/B, contains a BTB domain and kelch repeats and is expressed in primary neurons but not in primary glial cells. To examine the function of NRP/B in neurons, we analyzed the structure/function of the NRP/B-BTB domain and its role in neurite outgrowth. Based on three-dimensional modeling of NRP/B, we generated an NRP/B-BTB mutant containing three mutations in the conserved amino acids D47A, H60A and R61D that was termed BTB mutant A. BTB mutant A significantly reduced the dimerization of NRP/B compared to wild-type NRP/B. The NRP/B-BTB domain was required for nuclear localization and mediated the association of NRP/B with p110RB through the TR subdomain within the B pocket of p110RB. Overexpression of wild-type NRP/B and NRP/B-BTB domain significantly induced neurite outgrowth in PC12 cells and enhanced the G0-G1 cell population by approximately 23% compared to the control cells, whereas NRP/B-BTB mutant A reduced neurite outgrowth by 70-80%, and inhibited NRP/B-p110RB association. Single cell microinjection of NRP/B-specific antibodies also blocked the neurite outgrowth of PC12 cells upon NGF stimulation. Interference of NRP/B expression by small interfering RNA (NRP/B-siRNA) inhibited neurite outgrowth and suppressed the NGF-induced outgrowth of neurites in PC12 cells. Additionally, p110RB phosphorylation at serine residue 795 was significantly reduced in PC12 cells treated with NRP/B siRNA compared to those treated with control GFP-siRNA, indicating that p110RB is a downstream target of NRP/B. Thus, the BTB domain of NRP/B regulates neurite outgrowth through its interaction with the TR subdomain within the B pocket of p110RB, and the conserved amino acids D47A, H60A and R61D within this domain of NRP/B are crucial residues for neurite extension in neuronal cells. These findings support a role for the BTB-domain of NRP/B as an important regulator of neuronal differentiation.