Abstract: This study investigated the toxic effects of abamectin on human neuroblastoma SH-SY5Y cells at the cellular and molecular levels. The MTT assay, Western blotting, single-cell gel electrophoresis (comet assay), fluorescent staining, and immunofluorescence staining were employed to examine abamectin-induced DNA damage, mitochondrial impairment, and mitochondria-mediated apoptosis. Results demonstrated that abamectin inhibited SH-SY5Y cell viability (IC₅₀ = 20 μg/mL) causing mitochondrial damage, including mitochondrial permeability transition pore opening and collapse of membrane potential, DNA damage, such as DNA single- and double-strand breaks, as well as mitochondria-mediated apoptosis. The results indicate that abamectin exerts cytotoxic effects on human SH-SY5Y cells by inducing DNA damage and mitochondria-mediated apoptosis, while abamectin-induced apoptosis suppresses the DNA damage repair process. These findings contribute to the environmental toxicological assessment of abamectin, reveal its potential safety risks to human health, and provide a theoretical basis for improving its safe application.