Evaluations of the toxicity of hazardous substances in foods and other products are essential. Metals are severely neurotoxic to humans. To date, neurotoxicity has mainly been examined in animal experiments using rodents; however, the importance of using in vitro systems has recently been reported. Although the brain also contains glial cells, in vitro assessments of neurotoxicity have mainly been performed using neurons. Therefore, we established a high-throughput evaluation system by creating a mixed culture system of microglia, a type of glial cell, and neurons using BV2 and Neuro2A cells. The mixed culture showed changes in gene expression compared to the monoculture. Furthermore, in examinations of the toxicities of various metals, some exhibited different toxicities in the mixed culture from those in the single culture. These results suggest that the conventional evaluation system using single cultures is insufficient and also that the use of glial cells to accurately assess neurotoxicity may be necessary.