Significance Capsules are critical virulence determinants for bacterial pathogens. They are composed of capsular polysaccharides (CPSs) with diverse structures, whose assembly on the cell surface is often powered by a conserved ABC transporter. Current capsule-assembly models include a contiguous trans-envelope channel directing nascent CPSs from the transporter to the cell surface. This conserved apparatus is an attractive target for antivirulence antimicrobial development. This work describes a CPS depolymerizing lyase enzyme found in the Burkholderiales and unique structural features that define its mechanism, CPS specificity, and evolution to function in the periplasm in a noncatabolic role. The activity of this enzyme provides evidence that CPS assembled in an ABC transporter-dependent system is exposed to periplasm during translocation to the cell surface.