In the mammalian brain, alternative pre-mRNA splicing is a fundamental mechanism that modifies neuronal function dynamically where secretion of different splice variants regulates neurogenesis, development, pathfinding, maintenance, migration, and synaptogenesis. Sequence-specific RNA-Binding Protein CPEB3 has distinctive isoform-distinct biochemical interactions and neuronal development assembly roles. Nonetheless, the mechanisms moderating splice isoform options remain unclear. To establish the modulatory trend of CPEB3, we cloned and excessively expressed CPEB3 in HT22 cells. We used RNA-seq to analyze CPEB3-regulated alternative splicing on control and CPEB3-overexpressing cells. Consequently, we used iRIP-seq to identify CPEB-binding targets. We additionally validated CPEB3-modulated genes using RT-qPCR. CPEB3 overexpression had insignificant effects on gene expression in HT22 cells. Notably, CPEB3 partially modulated differential gene splicing enhanced in the modulation of neural development, neuron cycle, neurotrophin, synapse, and specific development pathway, implying an alternative splicing regulatory mechanism associated with neurogenesis. Moreover, qRT-PCR verified the CPEB3-modulated transcription of neurogenesis genes LCN2 and NAV2, synaptogenesis gene CYLD, as well as neural development gene JADE1. Herein, we established that CPEB3 is a critical modulator of alternative splicing in neurogenesis, which remarkably enhances the current understanding of the CPEB3 mediated alternative pre-mRNA splicing.