In this study, cobalt neurotoxicity was investigated in human astrocytoma and neuroblastoma (SH-SY5Y) cells using proliferation assays coupled with LC–MS-based metabolomics and transcriptomics techniques. Cells were treated with a range of cobalt concentrations between 0 and 200 µM. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed cobalt cytotoxicity and decreased cell metabolism in a dose and time-dependent manner was observed by metabolomics analysis, in both cell lines. Metabolomic analysis also revealed several altered metabolites particularly those related to DNA deamination and methylation pathways. One of the increased metabolites was uracil which can be generated from DNA deamination or fragmentation of RNA. To investigate the origin of uracil, genomic DNA was isolated and analyzed by LC–MS. Interestingly, the source of uracil, which is uridine, increased significantly in the DNA of both cell lines. Additionally, the results of the qRT-PCR showed an increase in the expression of five genes Mlh1, Sirt2, MeCP2, UNG, and TDG in both cell lines. These genes are related to DNA strand breakage, hypoxia, methylation, and base excision repair. Overall, metabolomic analysis helped reveal the changes induced by cobalt in human neuronal-derived cell lines. These findings could unravel the effect of cobalt on the human brain.