The adverse effects of elevated temperatures on human health are becoming progressively severe. This research established a mouse model of cognitive dysfunction induced by heat stress to examine the impact of omega-3 PUFAs on the cognitive capabilities of heat-stressed mice. The study also aimed to elucidate the role and potential mechanisms of autophagy regulation in cognitive enhancement through omega-3 PUFAs interventions. Administration of omega-3 PUFAs ameliorated cognitive deficits in heat-stressed mice and increased brain concentrations of these fatty acids. Notably, omega-3 PUFAs significantly protected hippocampal neurons' morphology, quantity, and synaptic architecture in heat-stressed mice. Additionally, omega-3 PUFAs intake reduced the prevalence of damaged mitochondria in the hippocampus and mitigated oxidative harm. Further investigation revealed that heat stress induces autophagy. However, the autophagic process becomes dysfunctional, leading to impaired autophagic activity. Omega-3 PUFAs supplementation markedly augmented hippocampal autophagy in the heat-stressed mice. Moreover, heat stress upregulated the phosphorylation of the PI3K-Akt-mTOR pathway in both the mouse hippocampus and HT22 cells. In contrast, omega-3 PUFAs intake significantly diminished the phosphorylation levels within this pathway, alleviating the autophagic fusion barrier imposed by heat stress and promoting autophagic flux. The findings suggest that omega-3 PUFAs supplementation during heat stress may bolster autophagic function by inhibiting the phosphorylation of the PI3K-Akt-mTOR pathway. This modulation reduces structural and oxidative stress damage, ultimately enhancing cognitive function in mice subjected to heat stress.