Glioblastoma (GB) cells actively interact with the central nervous system (CNS) tumor microenvironment (TME). These interactions, particularly with neurons, require a better understanding. 3D tumor models replicating the human TME are needed to unravel pathological processes and to test novel treatments for efficacy and safety. We developed a novel 3D human coculture model for studying neuron-GB interactions. The model revealed both structural and functional interactions between cell types. Paracrine communication in the coculture model favored a tumor-supportive environment. Notably, cell-specific calcium signaling characteristics differed in cocultures compared to monocultures, highlighting the impact of interactions on cellular functionality in TME. The safety of a clinically used treatment, temozolomide, was tested in the 3D coculture model, and it selectively inhibited GB invasion while preserving neurons' morphology and functionality. The established model provides a tool for dissecting the interactions within the TME and testing the efficacy and safety of novel treatments.