Polyphenol antioxidants are abundant in the diet and have the potential to prevent diseases caused by oxidative stress, including neurodegenerative and cardiovascular diseases, cancer, and stroke. Cellular studies are critical to understanding how polyphenol antioxidants can reduce oxidative stress and contribute to disease prevention. Many studies of polyphenol antioxidant mechanisms in E. coli and human cells have focused primarily on reactive oxygen species (ROS) scavenging and enzyme regulation, although iron-mediated hydroxyl radical generation and DNA damage is the primary cause of cell death for both prokaryotic and eukaryotic cells under oxidative stress. Recently, metal chelation has also been reported as a major contributor to cellular polyphenol antioxidant activity. Because ROS scavenging, enzyme regulation, and metal chelation mechanisms for polyphenol antioxidant activity are interrelated in vitro and in cellular systems, understanding the contribution of each mechanism to polyphenol antioxidant behavior is extremely challenging. Additionally, the wide variety of assays used to examine polyphenol antioxidant behavior makes comparing results between methods and between studies difficult. The complex relationships between ROS scavenging, enzyme regulation, and metal chelation mechanisms make selecting the appropriate cellular experiments vital to accurate mechanistic results, and incorrectly selected methods can lead to conflicting conclusions. Poor understanding of polyphenol cellular uptake also results in inconsistencies between in vitro, cellular, and in vivo studies. This chapter discusses and compares the evidence to support each of the three major mechanisms for polyphenol antioxidant activity in E. coli and human cells and describes the relationships between these three cellular mechanisms.