Microbiota of the human gut has been extensively shown to play a critical role in the health of the human host, with dysbiosis of the microbiota contributing to numerous diseases. Bacterial colonisation of the human gut is particularly dense and diverse, creating an environment where a wide variety of bacteria compete for resources. Bacteroidales species, which account for the predominant Gram-negative bacteria of the gut microbiota, have been shown to display bacterial competition at both an inter- and intraspecies level by production of various secreted antimicrobial protein toxins. We have recently discovered a toxin known as BcpT that mediates competition between strains of the species Bacteroides vulgatus or dorei via a currently unknown mechanism. Here we present the structure of BcpT solved by X-ray crystallography to a maximum resolution of 2.72 Å. BcpT lacks both sequence and structural similarity to any previously characterised proteins with the crystal structure revealing a novel fold. Furthermore, we have identified that BcpT must be proteolytically processed for activity via cleavage of two distinct sites located on surface-exposed loops of the protein, alongside identifying the putative endogenous proteases that may accomplish this activation. The crystal structure of proteolytically-activated BcpT mimics that of the pro-form. Further biophysical characterisation of the pro- and active-forms of BcpT, using small-angle X-ray scattering and hydrogen deuterium exchange mass spectrometry, further confirm that proteolytic cleavage achieves activation via small local changes in the protein rather than a drastic conformational change. Additionally, we show that BcpT binds the lipopolysaccharide (LPS) core on target bacterial cells. The study of natural antimicrobial proteins, such as described here for BcpT, provides insight into potential future alternatives to conventional antibiotics, which are currently plagued with the global crisis of antibiotic resistance.