Strict maintenance of brain cholesterol homeostasis is essential for neuronal functioning. Neuronal cholesterol is mostly synthesized in astrocytes, while the excess of this lipid is eliminated from the brain by its conversion into 24-S-hydroxycholesterol (24-OHC), via the neuronal specific enzyme CYP46A1. Although 24-OHC synthesis has been reported mainly in neurons, our results indicate that 24-OHC is produced at high rates by reactive astrocytes. The role of 24-OHC as a signaling molecule is controversial and has been implicated in Alzheimer's disease (AD). Therefore, we investigated the effect of 24-OHC in primary cultures of rat cortical neurons, analyzing: neuronal viability, density of synaptic contacts and its ability to induce APP synthesis. Our results show that minimal doses of 24-OHC significantly reduce the density of synaptic contacts, while higher concentrations affect neuronal viability, evidenced by an increase in pycnotic nuclei. In addition, exposure of primary neurons to 24-OHC increased APP synthesis from 1 μM, suggesting a possible predisposing role in AD at higher concentrations of this sterol. Our results suggest that 24-OHC would act as a mediator of neurotoxicity in astrogliosis with implications in AD.