Feed processing including milling techniques and thermal treatments, as well as feed particle size can significantly affect physiological processes in the digestive tract of laying hens and broiler chickens. Additionally, it has been shown that feeding coarse particles alters bacterial colonization in broiler chickens. However, the effect of feed processing on the intestinal bacteria of laying hens has rarely been investigated to date. We hypothesized that bacterial metabolites in the gastrointestinal tract of hens can be affected by feed processing, such as milling method, thermal treatment, and feed particle size.