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This study investigated the effects of bamboo vinegar (BV) supplementation on performance, eggshell quality, ileal microflora, and villus morphology in aged laying hens. A total of 320 hens (70 weeks old) were divided into 4 dietary treatments with 10 replicates of 8 hens each. They were fed a basal diet supplemented with BV at 0%, 0.2%, 0.4%, or 0.8% level until 86 weeks of age. The level of BV in the diet had no effect on egg performance, feed intake, or feed efficiency (p > 0.10). Damaged egg rate decreased in the hens fed the 0.4% and 0.8% BV diets during 79-86 weeks of age (p < 0.05). Eggshell thickness and eggshell strength were higher in the hens fed the 0.4% and 0.8% BV diets at weeks 82 and 86 compared to the controls. In the ileal content, population of Escherichia coli and Salmonella spp. decreased with 0.4% and 0.8% BV (p < 0.05). Villus height and areas of duodenum were higher in the hens fed the 0.4% and 0.8% BV diets (p < 0.05). The results indicate that a level of 0.4% BV in a layer's diet is sufficient for reducing pathogenic bacteria, stimulating intestinal functions and improving eggshell quality of laying hens in the late phase of production.