A high-fat diet (HFD) induces obesity and the associated increases in blood glucose and inflammation through changes in gut microbiota, endotoxemia, and increased gut permeability. To counteract this, researchers have suggested that the use of probiotics that suppress production of proinflammatory lipopolysaccharide (LPS). Here, we tested whether Lactobacillus sakei OK67, which inhibits gut microbiota LPS production selected from among the lactic acid bacteria isolated from kimchi, exerted antihypoglycemic or anti-inflammatory effects in HFD-fed mice. Mice were randomly divided into 2 groups and fed an HFD or a low-fat diet for 4 weeks. These groups were further subdivided; 1 subgroup was treated with L sakei OK67 and fed the experimental diet for 4.5 weeks, whereas the other subgroup was fed the experimental diet alone. L sakei OK67 treatment lowered HFD-elevated LPS levels in blood and colonic fluid and significantly decreased HFD-elevated fasting blood glucose levels and the area under the curve in an oral glucose tolerance test. L sakei OK67 treatment inhibited HFD-induced body and epididymal fat weight gains, suppressed HFD-induced tumor necrosis factor–α and interleukin-1β expression and nuclear factor–κB activation in the colon, and significantly increased HFD-suppressed interleukin-10 and tight junction protein expression in the colon. Oral administration of L sakei OK67 significantly downregulated HFD-induced expression of peroxisome proliferator-activated receptor γ, fatty acid synthase, and tumor necrosis factor–α in adipose tissue. In addition, L sakei OK67 treatment strongly inhibited nuclear factor–κB activation in LPS-stimulated peritoneal macrophages. We report that L sakei OK67 ameliorates HFD-induced hyperglycemia and obesity by reducing inflammation and increasing the expression of colon tight junction proteins in mice.