Yam yellow pigments (YP) are natural pigments formed during the storage of freshly cut yam () under certain conditions. The interaction of YP with calf thymus DNA (ctDNA) and its protective effect against DNA oxidative damage were investigated using multiple spectroscopic techniques, competitive binding experiments, viscosity measurements, and gel electrophoresis. Results showed that YP participated in intercalative binding with ctDNA. YP exhibited a protective effect against hydroxyl-induced DNA damage, which was attributed to the high hydroxyl radical scavenging activity of YP. Our findings improve our understanding of the mechanism of interaction between YP and ctDNA, and provide a theoretical basis for the application of YP in the food and drug industry.