<p>Double-negative T cells (DNTs; CD3⁺CD4⁻CD8⁻) have been implicated in immune regulation in autoimmune settings, but their relevance to reproductive tissue immune balance and inflammation-associated infertility remains unclear. Here, we define a population of TCRβ⁺NK1.1⁻ DNTs enriched in the mouse ovary and uterus and profiled its phenotype, tissue behavior, and function in inflammatory models associated with impaired fertility. In parallel, we performed RNA-seq on spleen- and thymus-derived NK1.1⁻ DNT and CD8⁺ T cell enriched population. Relative to CD8⁺ T cells, peripheral NK1.1⁻ DN T-cell-enriched populations displayed an activated, regulatory-like transcriptional profile with reduced <i>Cd8a</i>/<i>Cd8b1</i>, <i>Il7r</i>, and cytotoxic effector markers, alongside increased expression of <i>Pdcd1</i>, <i>Lag3</i>, <i>Tox</i>, and <i>Il10</i>. Ex vivo, FACS-sorted splenic DNTs produced low inflammatory cytokine output after CD3/CD28 stimulation and suppressed CD8⁺ T-cell proliferation primarily through contact-dependent mechanisms. In vivo, ovarian DNTs decreased following CD8-targeting depletion, and ovarian and uterine DNTs showed limited exchange in parabiosis. In chronic interferon-γ-driven inflammation (ARE^−/−) and zona pellucida 3-induced ovarian inflammation, reproductive tissues exhibited reduced DNT frequencies, a shift in the DNT: CD8⁺T ratio favoring CD8⁺ T cells and increased activated CD8⁺ phenotypes. Finally, adoptive transfer of ex vivo FACS-sorted wild-type DNTs into ARE^−/− females increased pregnancy frequency, supporting DNT-associated immunoregulation as a feature of inflammation-associated infertility.</p>