Abstract: Growing evidence has shown that sleep deprivation (SD) could result in Alzheimer’s disease (AD)-related pathological changes and cognitive decline. However, potential mechanisms remain obscure. Here, we demonstrated the existence of the microbiota-gut-brain axis in cognitive deficits resulting from chronic SD and revealed a potential pathway by which gut microbiota affected cognitive functioning in chronic SD. We found that, in addition to cognitive decline, gut microbiota dysbiosis occurred in chronic sleep-deprived mice, along with elevated NLRP3 inflammasome expression, GSK-3β activation, autophagy dysfunction, and tau hyperphosphorylation in the hippocampus. Colonization with the “SD microbiota” almost mimicked the pathological and behavioral abnormalities observed in chronic sleep-deprived mice. Strikingly, both deletion of NLRP3 using NLRP3-/- mice and specific knockdown of NLRP3 in the hippocampus restored autophagic flux, suppressed tau hyperphosphorylation, and ameliorated cognitive deficits induced by chronic SD, while the GSK-3β activity was not regulated by NLRP3 inflammasome in chronic SD. More intriguingly, we found that deletion of NLRP3 reversed NLRP3 inflammasome activation, autophagy deficits, and tau hyperphosphorylation induced by activation of GSK-3β in primary hippocampal neurons, suggesting GSK-3β as a regulator of NLRP3-mediated autophagy dysfunction plays a more significant role in promoting tau hyperphosphorylation. These findings identify gut microbiota dysbiosis as a contributor of chronic SD driving tau pathology via NLRP3-mediated autophagy dysfunction and ultimately causing cognitive deficits. And our findings highlight GSK-3β as a regulator of NLRP3-mediated autophagy dysfunction playing a more significant role in promoting tau hyperphosphorylation.