For many years, both preclinical and clinical studies have provided evidences to support the beneficial effects of ω-3 Polyunsaturated fatty acids (PUFAs), particularly Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) in the prevention of chronic diseases. However, recently, an increasing number of studies reported adverse or contradictory effects of ω-3 PUFAs on human health. While dose and experimental condition need to be considered when evaluating these effects, oxidation of PUFAs also serves as an important factor contributing to the inconsistent results. In fact, oxidation of PUFAs happens frequently during food processing and storage, cooking and even after food ingestion. The free radicals and metabolites generated from PUFA oxidation may adversely affect food quality and shelf life by producing off-flavors and reducing nutritional values. The impact of PUFA oxidation in human health is more complicated, depending on the concentration of products, disease background and targets. This review will introduce different types of PUFA oxidation, discuss its impact on food quality and human health and provide some thoughts for the future research directions.