The ankyrin repeat is one of the most common protein motifs and is involved in protein-protein interactions. It consists of 33 residues that assume a β-hairpin helix-loop-helix fold. Mutagenesis and kinetic experiments (Φ-value analysis of the folding transition state) have shown that the tumor suppressor p16^INK4a, a four-repeat protein, unfolds sequentially starting from the two N-terminal repeats. Here, the flexibility of p16^INK4a at room temperature and its unfolding mechanism at high temperature have been investigated by multiple molecular dynamics runs in explicit water for a total simulation time of 0.65 μs. The transition state ensemble (TSE) of p16^INK4a was identified by monitoring both the deviation from the experimental Φ values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid-body displacement of the first repeat involving both a rotation and translation is observed in all molecular dynamics simulations at high temperature. The Trp₁₅, Pro₇₅, and Ala₇₆ side-chains are more buried in the TSE than the native state. The sequential unfolding starting at the second repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding trajectories reveal the presence of two on-pathway intermediates with partial α-helical structure. Finally, on the basis of the available experimental and simulation results we suggest that in modular proteins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect.