Complex I is the largest and most complicated enzyme of the respiratory chain. The enzyme is found in the inner mitochondrial membrane of eukaryotes and the plasma membrane of many prokaryotes, where it functions to oxidize NADH and reduce ubiquinone. This reaction is an essential step in the process of oxidative phosphorylation, which generates ATP from the energy stored in nutrients. In this review, we aim to present the current understanding of the enzyme, covering its structure, function, and evolution. As we will see, complex I is a dynamic and ancient enzyme that has evolved many times in response to different challenges and opportunities.