Development of pro-neurogenic therapies for compensating the neuronal loss and restoration of degenerated synaptic networks in neurodegenerative diseases has been difficult to achieve, which calls for new strategies to promote adult neurogenesis and counteract neurodegeneration. BRI2 is a ubiquitously expressed transmembrane protein that undergoes proteolytic processing, resulting in the formation of several secreted peptides. The precise physiological function of BRI2 remains elusive, however preliminary results suggested an increase in BRI2 levels during neuronal development, and a strong phenotypic association between BRI2’s processing and neuronal differentiation. Thus, our aim is to explore the contribution of BRI2 proteolytic fragments for neuronal differentiation. In this work, BRI2 expression and processing was evaluated in relevant cellular models (differentiated SH-S5Y5 cells and primary neuronal cultures-PNC) by western blotting and immunocytochemistry. Further, pharmacological modulation of BRI2 processing was achieved using GI254023X and (Z-LL)2 ketone, an ADAM10 and SPLL2b specific inhibitors, respectively. The effects of these compounds were evaluated by performing a morphometric analysis to characterize cells differentiation phenotypes. Our results reveal that BRI2 expression and processing increases throughout SH-SY5Y differentiation and in the early stages of PNC differentiation. Moreover, inhibition of BRI2 processing impacts the number and length of neurites/dendrites supporting a role for BRI2 fragments in neuronal differentiation. With this work, we expect to contribute to expand the knowledge on the CNS by deciphering the neuritogenic nature of BRI2 and its fragments, which hopefully will contribute to a deeper understanding of BRI2 as a therapeutic target to neurological conditions characterized by neuronal loss.