Sleep disorders and deficiencies can up-regulate pro-inflammatory mediators, but their potential adverse impact on ovarian function remains not fully understood. In this study, we investigated the immunoregulatory mechanisms underlying sleep deprivation (SD)-induced ovarian damage and the effects on follicular development. We found that SD disrupted follicular development and compromised early embryonic developmental potential in female mice. Notably, the use of the anti-inflammatory drug acetaminophen can effectively alleviate the adverse effect. Bulk and single-cell RNA sequencing of ovarian tissues revealed a notable up-regulation of neutrophil-associated genes, including S100a8, S100a9, the macrophage M1 polarization gene set, and pyroptosis-related genes in granulosa, immune, and mesothelial cells. Further experiments conducted both in vivo and in vitro verified that S100A8/A9 activates the TLR4/Myd88/NF-κB pathway, which induces the polarization of M1 macrophages and leads to pyroptosis of granulosa cells. Collectively, this study provides molecular insight into strategies for fertility preservation in individuals experiencing sleep deprivation, including those with occupational circadian rhythm disruptions.