Neural tissue-specific epidermal growth factor-like repeat domain-containing protein (NELL2) was previously found to play an important role in nerve growth, neural differentiation, neural elasticity, synaptic transport, and vesicle release. In this study, we examined the effect of NELL2 on gonadotropin-releasing hormone (GnRH) neurons and the initiation of puberty in female rats. We studied changes in NELL2 mRNA and protein expression at different stages of sexual development (postnatal days 30, 35, and 45) in female rats to determine the impact of NELL2 on GnRH mRNA expression. We also investigated the influence on the vulva-opening age by inhibiting NELL2 mRNA expression through lentiviral vector-mediated RNA interference. The intraventricular administration of an NELL2-interfering virus reduced NELL2 and GnRH expression at multiple stages of sexual development and delayed the age of vulva-opening in female rats. These results demonstrate that lentiviral-mediated RNA interference technology can be used for targeted regulation of sexual development in vivo. In addition, we found that NELL2 regulated the initiation of puberty in female rats.