Background and Aims: Gut dysbiosis, an imbalance of gut microbiota, is associated with host inflammation due to the translocation of Gram-negative bacteria-derived lipopolysaccharide (LPS), resulting from increased gut permeability. Inflammation plays a key role in the pathophysiology of ischemic stroke. Here, we investigated the effect of gut dysbiosis on acute ischemic brain injury. Methods: Fecal bacteria were analyzed via 16S and 23S rRNA-targeted quantitative reverse transcription-polymerase chain reaction and the outcome of focal cerebral ischemia was examined in lean littermates (db/+), type 2 diabetic mice (db/db), and db/db mice with oral administration of the non-absorbable antibiotic polymyxin B (db/db+PL-B) (n=5-9/group). Results: Mice from the db/db+PL-B group had selectively attenuated fecal bacterial count of the Gram-negative bacteria Enterobacteriaceae (p<0.01), and reduced plasma LPS levels (p<0.05) and gut permeability (p<0.05) without a change in plasma glucose levels compared to those in db/db mice. Polymyxin B was not detected in the blood of db/db+PL-B mice. In addition, antibiotic pre-treatment significantly reduced infarct size (p<0.05), blood-brain barrier leakage (p<0.05), inflammatory reactions in the brain, such as IL-6 (p<0.01), IL-1β (p<0.01), and TNF-α (p<0.05), improved neurological function (p<0.05), and survival rates (p<0.05) following cerebral ischemia. Furthermore, db/db + PL-B mice had significantly reduced LPS (p<0.05) and expression of TLR4 (p<0.01), the LPS receptor, and inflammatory cytokine levels, such as IL-6 (p<0.01), IL-1β(p<0.01), and TNF-α (p<0.05), in the ischemic hemisphere. Conclusions: These findings suggest that LPS-induced inflammation may be a mechanism that links gut dysbiosis to ischemic cerebral injury, and that modulation of gut microbiota may be a potential therapeutic strategy to improve the stroke outcome.