Mitochondria are ubiquitous organelles in eukaryotic cells whose primary function is to generate energy supplies in the form of ATP through oxidative phosphorylation. As the entry point for most electrons into the respiratory chain, NADH:ubiquinone oxidoreductase, or complex I, is the largest and least understood component of the mitochondrial oxidative phosphorylation system. Substantial progress has been made in recent years in understanding its subunit composition, its assembly, the interaction among complex I and other respiratory components, and its role in oxidative stress and apoptosis. This review provides an updated overview of the structure of complex I, as well as its cellular functions, and discusses the implication of complex I dysfunction in various human diseases.