A newly discovered vertebrate assemblage is reported from the Upper Pennsylvanian (mid- to upper Kasimovian) Cohn Coal Member of the Mattoon Formation of southeast Illinois, United States. Teeth, scales, and spines of xenacanth (Dicentrodus, Orthacanthus, Triodus, Xenacanthus) and euselachian (Sphenacanthus) sharks dominate the assemblage. Less common are the teeth, scales, and centra of holocephalan (Helodus) and actinopterygian fishes, together with rare tetrapod (mainly pelycosaur) phalanges and centra. The assemblage occurs within a broad, shallow channel incised into a prominent Vertisol. The channel is interpreted as having been cut during a seasonally dry glacial phase when sea level was low, but filled during a subsequent transgression triggered by deglaciation. We interpret this as a brackish water (estuarine) assemblage, based on the co-occurrence of the vertebrate material with spirorbids (putative microconchids) and paleoecological inferences gleaned from a critical analysis of the literature dealing with Pennsylvanian fish ecology. This interpretation is broadly consistent with taphonomic data and the results of ⁸⁷Sr/⁸⁶Sr isotope analysis of shark material. The pelycosaur material may have been reworked from the lowstand Vertisol, however, and these animals occupied dryland niches that developed during glacial phases.