OBJECTIVE: Ferroptosis, a regulated cell death pathway driven by lipid peroxidation and iron overload, is implicated in neuronal injury caused by hypoxia/reoxygenation (H/R). Caffeine, a widely consumed psychoactive compound, has shown neuroprotective effects in various central nervous system disorders, but its role in regulating ferroptosis remains unclear. This study investigates the neuroprotective effects of caffeine on ferroptosis and its regulation of ACSL4, a key ferroptosis-related protein. METHODS: Molecular docking was performed to evaluate the interaction between caffeine and ferroptosis-related proteins ACSL4 and GPX4. HT-22 cells were subjected to H/R to establish an in vitro injury model, followed by treatment with caffeine at varying concentrations. ACSL4 was silenced or overexpressed to explore its role in caffeine-mediated ferroptosis regulation. Cell viability, inflammatory cytokines, ferroptosis markers, and mitochondrial function were assessed. RESULTS: Molecular docking revealed favorable binding affinities of caffeine with ACSL4 (-5.6 kcal/mol) and GPX4 (-4.6 kcal/mol). Caffeine treatment dose-dependently improved cell viability, reduced TNF-α, IL-1β, and IL-6 levels, and inhibited ferroptosis by downregulating ACSL4 and upregulating GPX4. Overexpression of ACSL4 reversed these protective effects, increasing lipid peroxidation markers (iron, Fe2+, ROS, and MDA) and reducing GSH levels and mitochondrial membrane potential. Conversely, silencing ACSL4 enhanced caffeine's protective effects, confirming its role as a critical target of caffeine-mediated ferroptosis inhibition. CONCLUSION: Caffeine protects against H/R-induced neuronal injury by regulating ACSL4-mediated ferroptosis, reducing oxidative stress and inflammation. These findings highlight ACSL4 as a therapeutic target and provide mechanistic insights into caffeine's neuroprotective potential.