The molecular mechanisms governing neuronal morphogenesis remain largely unknown. Among these, neuronal membrane glycoprotein GPM6a is known to promote differentiation and synaptogenesis, and its dysregulation is linked to neuropsychiatric disorders. Given that the extracellular loops (ECs) of GPM6a are critical to its function, previous studies from our laboratory identified that the cell adhesion molecule neuroplastin (NPTN) co-immunoprecipitates with GPM6a using its ECs as bait. This study investigates the functional association between GPM6a and NPTN in hippocampal neurons and cell lines. Endogenous NPTN and GPM6a colocalize at the neuronal membrane across various developmental stages. Co-overexpression of both proteins inhibited neurite extension, reduced the number of neurites per cell, and decreased filopodia formation, indicating a non-cooperative interaction during neuronal development. In HEK293 cells, the NPTN ectodomain and GPM6a-ECs interacted in a trans configuration, inducing cell aggregation. This aggregation was inhibited by adding a calcium chelator (EGTA) or GPM6a-neutralizing monoclonal antibodies. Importantly, an NPTN isoform lacking an IgG domain (NPTN55) did not aggregate with GPM6a, nor did a GPM6a mutant deficient in EC2 folding aggregate with wild-type NPTN. Collectively, these findings suggest that co-overexpression of GPM6a and NPTN acts antagonistically and support a functional interaction between GPM6a and NP65 via their extracellular domains.