A significant body of evidence suggests that the marine environment remained largely anoxic throughout most of the Precambrian. In contrast, the oxygenation history of terrestrial aquatic environments has received little attention, despite the significance of such settings for early eukaryote evolution. To address this, we provide here a geochemical and isotopic assessment of sediments from the late Mesoproterozoic Nonesuch Formation of central North America. We utilize rhenium-osmium (Re-Os) geochronology to yield a depositional age of 1078 ± 24 Ma, while Os isotope compositions support existing evidence for a lacustrine setting. Fe-S-C systematics suggest that the Nonesuch Formation was deposited from an anoxic Fe-rich (ferruginous) water column. Thus, similar to the marine realm, anoxia persisted in terrestrial aquatic environments in the Middle to Late Proterozoic, but sulfidic water column conditions were not ubiquitous. Our data suggest that oxygenation of the terrestrial realm was not pervasive at this time and may not have preceded oxygenation of the marine environment, signifying a major requirement for further investigation of links between the oxygenation state of terrestrial aquatic environments and eukaryote evolution.