Zinc deficiency is predicted to affect over 1 billion (17%) of the world’s population, including 12% of the U.S. population. Over 3000 human proteins bind zinc, where zinc plays vital biochemical and structural roles in cellular and organ function. Widely utilized biomarkers of zinc nutritional status lack sensitivity and specificity to assess zinc intervention impact due to the ubiquitous nature of zinc in human biological systems. Biofortification to deliver increased bioavailable zinc through staple crops, either through increasing zinc or crop-derived bioactive content, or a combination thereof, represents a promising strategy to complement existing zinc intervention efforts. This dissertation presents the utilization of a panel of predictive indices to assess zinc status reliably and accurately and further investigates the utility of novel zinc status biomarkers. The role of biofortified staple crops and food ingredients in altering intestinal functionality and the gut microbiome as it relates to intestinal zinc absorption and status is also presented. Chapter 1 summarizes zinc essentiality in human health, intestinal zinc absorption, zinc biomarkers, and zinc deficiency alleviation strategies. Chapter 2 systematically reviews literature on gut microbiome composition and function as it relates to dietary zinc and zinc status and assesses the applicability of the gut microbiome as a potential zinc status biomarker. Chapter 3 evaluates potential negative implications associated with food additive nanoparticle consumption on intestinal functionality and morphology in vivo and thus essential nutrient (zinc) absorption and bioavailability. Chapter 4 examines the beneficial and nutritional effects of genistein (plant-origin bioactive) in potentially improving mineral (zinc) dietary bioavailability, intestinal functionality, morphology, and altering gut microbiome composition in vivo. Chapter 5 focuses on understanding nicotianamine (mineral transporter naturally present in plants) enhancement as a potential strategy for zinc biofortification via in vivo screening of three varieties of nicotianamine-biofortified wheat on improving mineral (zinc) bioavailability and intestinal functionality and morphology. Chapter 6 focuses on development and validation of the Zinc Status Index concept, a statistical model based on a panel of biochemical markers reactive to dietary zinc manipulation. To conclude, Chapter 7 highlights significant findings in Chapters 2–6 and suggests future work for each.