Botulinum neurotoxins (BoNTs, serotypes A-G), the most toxic substances known to mankind, cause flaccid muscle paralysis by blocking acetylcholine release at nerve-muscle junctions through a very specific and exclusive endopeptidase activity against SNARE proteins of presynaptic exocytosis machinery. We have examined polypeptide folding of the endopeptidase moiety of BoNT/A (the light chain) under conditions of its optimal enzymatic activity, and have found that one of its stable conformational states is a molten-globule, which retains over 40% of its optimal enzyme activity. More importantly, we have discovered that the light chain acquires a novel Pre-Imminent Molten-globule Enzyme (PRIME) conformation at the physiologically relevant temperature, 37 °C. The PRIME form also exhibits the maximum endopeptidase activity against its intracellular substrate, SNAP-25. These findings not only open new avenues to design effective diagnostics and antidotes against botulism, but also provide new information on enzymatically active molten-globule or molten-globule like structures.