The cognitive impairments resulting from insufficient sleep have emerged as a significant public health concern in contemporary society, lacking effective treatments due to the absence of elucidation regarding its molecular mechanism. This study aimed to investigate the efficacy of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against cognitive deficits in a mouse model of chronic sleep deprivation (CSD), and to elucidate the underlying mechanisms. CSD mice displayed significant cognitive deficits in the Morris water maze task with obvious neuronal damage. We observed that dysregulation of iron transport proteins (IRP1, TfR1, DMT1, and FPN1) in CSD mice resulted in iron deposition in the cortex. Subsequent assessment revealed upregulation of mitophagy and neuronal lipid peroxidation and downregulation of proteins in the mitochondrial respiratory chain complexes (Ⅰ-Ⅴ). Further molecular analysis revealed inhibition of the Sirt3-Nrf2 pathway and its downstream antioxidant molecules (e.g., GPX4, HO-1, SOD2, and NQO1). In contrast, dietary supplementation with DHA or EPA alleviated CSD-induced cognitive impairment by inhibiting neuronal ferroptosis through regulating iron-mitophagy homeostasis. Notably, EPA showed superior efficiency in ameliorating cognitive impairment, potentially due to its stronger interaction with Sirt3 compared to DHA.