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Gut microbiota are known to play an important role in obesity. <i>Enterobacter cloacae</i>, a Gram-negative bacterium, has been considered a pathogenic bacterium related to obesity in the gut. In this study, we established an obesity model of <i>C. elegans</i> by feeding <i>E. cloacae</i> combined with a high glucose diet (HGD), which significantly induced lipid accumulation. An anti-lipid mechanism study revealed that the fatty acid composition and the expression level of fat metabolism-related genes were altered by feeding <i>E. cloacae</i> to <i>C. elegans</i> under HGD conditions. Lactic acid bacteria that showed antagonistic activity against <i>E. cloacae</i> were used to screen anti-obesity candidates in this model. Among them, <i>L. pentosus</i> MJM60383 (MJM60383) showed good antagonistic activity. <i>C. eleans</i> fed with MJM60383 significantly reduced lipid accumulation and triglyceride content. The ratio of C18:1Δ9/C18:0 was also changed in <i>C. elegans</i> by feeding MJM60383. In addition, the expression level of genes related to fatty acid synthesis was significantly decreased and the genes related to fatty acid β-oxidation were up-regulated by feeding MJM60383. Moreover, MJM60383 also exhibited a high adhesive ability to Caco-2 cells and colonized the gut of <i>C. elegans</i>. Thus, <i>L. pentosus</i> MJM60383 can be a promising candidate for anti-obesity probiotics. To the best of our knowledge, this is the first report that uses <i>E. cloacae</i> combined with a high-glucose diet to study the interactions between individual pathogens and probiotics in <i>C. elegans</i>.