The homotrimeric ligand tumor necrosis factor alpha (TNF alpha) is a key cytokine and immune regulator; however, when deregulated, it leads to several major chronic inflammatory diseases. Perturbation of the protein-protein interface has proven to be an efficient strategy to inactivate TNF alpha, but the atomic-resolution mechanism of its inactivation remains poorly understood. Here, we 90 probe the solution structure and dynamics of active and inactive TNF alpha using NMR spectroscopy. The data reveal that TNF alpha undergoes motions on different time scales. Furthermore, by site-directed mutagenesis of residues at the trimerization interface and by targeting the interface with a low molecular weight inhibitor, we show that TNF alpha retains its overall structure and trimeric state. However, upon perturbation, TNF alpha exhibits increased conformational dynamics spanning from the trimerization interface to the regions mediating receptor binding. These findings provide novel insights into the inactivation mechanism of TNF alpha and the basis for strategies to target TNF alpha activity.