The widespread use of atrazine (ATR), a commonly applied herbicide, has raised growing concerns about its neurotoxic effects. However, the underlying molecular mechanisms remain poorly understood. In this study, we demonstrate that ATR disrupts hippocampal function by inducing Drp1-mediated mitochondrial fission. Using both in vivo and in vitro tests, we show that ATR exposure leads to mitochondrial swelling, cristae loss, and fragmentation in hippocampal neurons, correlating with impaired spatial learning and memory. ATR significantly increases Ser616-Drp1 phosphorylation, promoting excessive mitochondrial fission and exacerbating neuronal damage. In contrast, the Drp1 inhibitor Mdivi-1 effectively restores mitochondrial integrity, mitigates mitochondrial membrane potential loss, and alleviates neurotoxicity. Interestingly, ATR exposure results in a non-linear response in the expression of mitochondrial regulatory genes, suggesting complex dose-dependent effects. These findings provide novel insights into the role of mitochondrial dysfunction in ATR-induced cognitive impairment and underscore the importance of Drp1-mediated fission in herbicide neurotoxicity. Our study highlights the need for further investigation into the long-term effects of ATR exposure and suggests that targeting mitochondrial dynamics may offer a promising therapeutic strategy for ATR-induced neuronal dysfunction.