Antibody-mediated neutralization of virus infectivity plays a key role in host defence and protection against re-infection, and is an essential component of effective vaccine strategies. However, the structural basis of the different mechanisms by which neutralization occurs is poorly understood. We have generated a panel of mouse monoclonal antibodies (mAbs) to the dengue virus (DENV) envelope (E) protein domain III (EDIII), and have performed structural and functional characterization of two that neutralize all four DENV serotypes (3E31 and 2D73). The target of these antibodies, the dengue virus E protein, is responsible for host-cell receptor binding, as well as cellular entry following low pH-triggered conformational changes that drive membrane fusion. We have shown that one of these cross-reactive neutralizing antibodies, 2D73, can trigger premature fusion in a pH-independent manner, while the other, 3E31, inhibits fusion. The two mAbs recognize overlapping but distinct epitopes and their structural characterization provides insight into a new mechanism of enveloped virus neutralization via premature triggering of the fusion protein, laying the foundation for new strategies for vaccine and therapeutics development.