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The experiment was to determine the chronic effects of two transgenic maize lines that contained the <i>mCry1Ac</i> gene from the <i>Bacillus thuringiensis</i> strain (BT) and the <i>maroACC</i> gene from <i>Agrobacterium tumefaciens</i> strain (CC), respectively, on ileal microbiota of laying hens. Seventy-two laying hens were randomly assigned to one of the three dietary treatments for 12 weeks, as follows: (1) nontransgenic near-isoline maize-based diet (CT diet), (2) BT maize-based diet (BT diet), and (3) CC maize-based diet (CC diet). Ileum histological examination did not indicate a chronic effect of two transgenic maize diets. Few differences were observed in any bacterial taxa among the treatments that used high-throughput 16S rRNA gene sequencing. The only differences that were observed for bacterial genera were that <i>Bifidobacterium</i> belong within the <i>Bifidobacteriaceae</i> family tended to be greater (<i>p</i> = 0.114) abundant in hens fed the transgenic maize-based diet than in hens fed the CT diet. Birds that consumed the CC maize diet tended to have less abundance (<i>p</i> = 0.135) of <i>Enterobacteriaceae</i> family in the ileum than those that consumed the CT maize diet. These results indicate the lack of adverse effects of the BT maize and the CC maize lines on the ileal microbiota of hens for long term and provide important data regarding biosafety assessment of the transgenic maize lines.