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Rich in abundant polyphenols and dietary fiber, blueberry pomace is still a potential material for gut health. This study aimed to reuse blueberry pomace by lactic acid bacteria fermentation and evaluate the colonic barrier-improving functions of Lactobacillus casei-fermented blueberry pomace (FBP). Male C57BL/6 mice were fed with control or high fat diets. The changes of oxidative stress, inflammation, microbiota, short chain fatty acids (SCFAs), SCFAs receptors and barrier-related proteins in mice colon were evaluated. FBP attenuated the high fat diet-induced oxidative and inflammatory status in colon. Abundance of Bifidobacterium, Lactobacillus, Ruminococcus, Akkermansia and butyrate-producing bacteria, SCFAs concentration, expression of SCFAs receptors and regulation of barrier-related proteins in high fat diet mice were elevated through supplying FBP. These FBP-associated beneficial changes in high fat diet mice were mediated by the MAPK-NF-κB-MLCK signaling pathway. FBP shows colonic barrier-improving functions in high fat diet mice.