Uracil (U) can be found in DNA as a mismatch paired either to adenine (A) or to guanine (G). Removal of U from DNA is performed by a class of enzymes known as uracil–DNA–glycosylases (UDG). Recent studies suggest that recognition of U–A and U–G mismatches by UDG takes place via an extra-helical mechanism. In this work, we use molecular dynamics simulations to analyze the structure, dynamics and open base pair kinetics of U–A base pairs relative to their natural T–A counterpart in 12 dodecamers. Our results show that the presence of U does not alter the local conformation of B-DNA. Breathing dynamics and base pair closing kinetics are only weakly dependent on the presence of U versus T, with open T–A and U–A pairs lifetimes in the nanosecond timescale. Additionally, we observed spontaneous base flipping in U–A pairs. We analyze the structure and dynamics for this event and compare the results to available crystallographic data of open base pair conformations. Our results are in agreement with both structural and kinetic data derived from NMR imino proton exchange measurements, providing the first detailed description at the molecular level of elusive events such as spontaneous base pair opening and flipping in mismatched U–A sequences in DNA. Based on these results, we propose that base pair flipping can occur spontaneously at room temperature via a 3-step mechanism with an open base pair intermediate. Implications for the molecular basis of U recognition by UDG are discussed.