UNLABELLED: The addition of trace minerals into the diet of lactating cows frequently exceeds national recommendations for industry practices. However, the presence of certain heavy metals, such as zinc and copper, has been shown to exert selection pressure on the gut microbiota, favoring metal resistance and potential co-selection for antimicrobial resistance. To determine whether oversupplementation of dietary zinc alters the gut microbiota of dairy cattle, a cross-over design was used to feed either recommended or surplus levels of dietary zinc (0.89×
high mineral diet) compared to the recommended levels (control diet). Rumen, duodenum, and fecal samples were collected to analyze the 16S rRNA microbial community for diversity and relative abundance, with a greater focus on the IMPORTANCE: The addition of trace minerals into the diet of lactating cows, at levels exceeding national recommendations, is a common industry practice. However, there are new concerns as the presence of certain heavy metals, such as zinc, has been shown to exert selection pressure on the gut microbiota, favoring metal resistance and potential co-selection for antimicrobial resistance. We evaluated how the addition of zinc in the diet of lactating cows affects the bacterial community's relative abundance and diversity, with a focus on the