Metronidazole-induced neurotoxicity is a rising challenge in managing susceptible infections. The mechanisms involved in metronidazole-induced neurotoxicity are not fully unraveled. This study aimed to explore the effect of metronidazole on iron homeostasis in SH-SY5Y neuroblastoma cells. Confluent SH-SY5Y neuroblastoma cells were treated with different concentrations of 1.0, 10, 25, 50, 100, and 250 µM of metronidazole only or in combination with 20 µM iron. DMSO or culture media was used as control. Viability and ferritin assays were conducted on the treated cells. The treatments were for 24 hours, 48 hours, and 72 hours, respectively. In the viability assay, doses of metronidazole reduced the viability of SH-SY5Y neuroblastoma cells in a time and concentration-dependent manner. After 24-hour treatment, 250 µM metronidazole significantly reduced cell viability while 50 µM, 100 µM and 250 µM metronidazole reduced considerably viability only after 48-hour and 72-hour compared with control. Different doses of metronidazole 50 µM, 100 µM, and 250 µM in 20 µM iron reduced viability in a time-dependent manner in all the test periods. Metronidazole also induced a time- and concentration-dependent increase (p<0.05) in cellular iron uptake in the 48-hour and 72-hour treated cells in concentrations above 25 µM metronidazole. It is concluded that metronidazole induces a time and concentration-dependent iron overload and consequent cell death in SH-SY5Y neuroblastoma cells and this may contribute to the mechanism of metronidazole-induced neurotoxicity.