This study investigated the antidepressant activity and underlying mechanism of Eucommia ulmoides male flower water extract (EUMF) based on zebrafish model and molecular dynamics simulation. The ameliorative effects of EUMF on depressive-like behaviors in zebrafish were evaluated through a novel tank test. Subsequently, brain tissues were collected for qPCR analysis to measure the expression of depression-related and oxidative stress-associated genes. The levels of reactive oxygen species (ROS) in the brain were determined. The transcriptomic sequencing was conducted to explore the molecular mechanisms. The identified genes involved in neurotransmitter transporter activity were further validated by qPCR. ELISA method was used to assess the impact of EUMF on brain neurotransmitter levels. Additionally, molecular dynamics simulation was performed to analyze the interaction between key active compounds in EUMF and their potential protein targets. The results showed that EUMF significantly alleviated depressive-like behaviors and normalized the expression of depression marker genes, including prolactin (prl), melanocortin 2 receptor (mc2r), and hypocretin neuropeptide precursor (hcrt), indicating its antidepressant potential. Mechanistically, EUMF reduced ROS levels in the brain and restored the expression of oxidative stress-related genes such as superoxide dismutase 1 (sod1), sod2, glutathione S-transferase 2 (gst2), glutathione peroxidase 1a (gpx1a), and gpx4a. This reduction in oxidative stress might protect against neuronal damage and preserve the function of γ-aminobutyric acid (GABA) transporters. This was evidenced by the upregulation of solute carrier family 6 member 6b (slc6a6b) and solute carrier family 6 member 11b (slc6a11b), which enhanced GABA levels in the brain and maintained the ratio of 5-hydroxytryptamine/5-hydroxyindoleacetic acid (5-HT/5-HIAA) homeostasis, contributing to the antidepressant effect. Molecular docking and dynamics simulation further revealed the interactions between the main active components of EUMF and SLC6A6 and SLC6A11 proteins. In conclusion, EUMF reduced oxidative stress in the brain, maintaining GABA levels and stabilizing the 5-HT/5-HIAA balance, thus exerting antidepressant effects.