INTRODUCTION: Ischemic stroke (IS) represents a significant global health challenge, characterized by elevated morbidity and mortality rates, largely driven by inflammatory responses. Double-negative T cells (DNTs), a distinct subset of T cells lacking both CD4 and CD8 markers, have been implicated in the pathogenesis of IS, exhibiting potentially dual roles. However, the precise functional contributions of DNTs in this context remain poorly understood. METHODS: In this study, we investigated the role of DNTs during the acute phase of IS and assessed the influence of Huang-Lian-Jie-Du Decoction (HLJD), a traditional Chinese medicinal formula, on these cells. Using single-cell transcriptomics, we identified two distinct subtypes of DNTs: an activated, cytotoxic phenotype (Kill+) and a resting, immunosuppressive phenotype (Kill-). RESULTS: Our findings indicate that HLJD treatment modulates the balance between these DNT subtypes, specifically reducing the proportion of cytotoxic DNTs while promoting an increase in immunosuppressive DNTs. This shift was associated with a reduction in immune cell infiltration and inflammation within the brain tissue, potentially mitigating neuronal damage. DISCUSSION: These results suggest that HLJD exerts neuroprotective effects in IS by modulating the activity and distribution of DNT cells, offering valuable insights into the therapeutic potential of traditional Chinese medicine for the treatment of IS. Further studies are required to elucidate the mechanisms underlying DNT-mediated immune responses in IS and to explore the broader applications of HLJD in other neuroinflammatory conditions.