Micronutrient deficiencies, including iron (Fe) and zinc (Zn), are a significant global public health concern. Biofortification is a cost-effective and sustainable approach to delivering micronutrients through staple foods to populations with limited access to a diverse diet and nutrition. This study evaluated the effect of transgenic biofortified rice (Oryza sativa) from the Philippines and Bangladesh on the duodenal brush border membrane key protein expression and cecal bacterial population in vivo (Gallus gallus). Significant differences in DMT1 were found between treatments P2 (Zn/Fe transgenic) and P3 (control) in the Philippine rice and between B1 (Fe/Zn transgenic) and B5 (Zn enriched) in the Bangladeshi diet. In the abundance of Bifidobacterium and Lactobacillus spp., P1 had the highest values in the Philippine group, while B1 showed a significantly higher density of Bifidobacterium spp. in the Bangladeshi diet. Thus, this study suggested that biofortified rice may affect iron absorption and the gut microbiome.