Abstract: This study aimed to assess the neurotoxic effects of the novel psychoactive substance 4-hydroxy-N-methyl-N-isopropyltryptamine (4-HO-MiPT) on brain. By employing zebrafish as a model organism, the study applied a multifaceted approach, including the recording and analysis of spontaneous behavioral activities, identification of brain metabolic products via ultra-high performance liquid chromatography coupled with quadrupole-Orbitrap high resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS), and examination of changes in the transcriptional level of target genes. Following the injection of 4-HO-MiPT, zebrafish exhibites a pronounced reduction in movement speed and range, indicating a significant alteration in locomotor activity. The subsequent analysis of brain metabolic products finds 37 differential metabolites, including 7 elevated and 30 reduced ones, alongside notable alterations in 6 metabolic pathways. Moreover, the examination of transcriptional level of key genes in zebrafish brain underscores a discernible decrease in transcriptional level of genes associated with the nervous system, implying a substantial impact on neuronal function and synaptic transmission. The experimental findings substantiate the neurotoxic effects of 4-HO-MiPT on the zebrafish brain, shedding light on its deleterious repercussions on both the nervous and immune systems. Furthermore, the perturbations observed in metabolic processes and behavioral responses emphasize the profound impact of 4-HO-MiPT exposure on fundamental physiological functions. The identification of potential carcinogenicity associated with 4-HO-MiPT further underscores the urgent need for comprehensive risk assessment and regulatory scrutiny. This study represents a crucial step towards unraveling the intricate neurotoxic mechanisms underlying 4-HO-MiPT exposure. The research has far-reaching implications, not only in advancing our understanding of the adverse health effects associated with novel psychoactive substances, but also in guiding the development of targeted interventions and regulatory strategies aiming at safeguarding public health and well-being. Continued exploration into the long-term consequences of 4-HO-MiPT exposure, alongside comprehensive toxicological evaluations, holds promise in mitigating its potential hazards and ensuring the safety of individuals and communities exposed to this emerging threat.