The mid-Proterozoic (1.8–0.8 billion years ago, Ga) is generally considered to have been an interval of relative stasis in terms of environmental and biological evolution. Indeed, in terms of ocean redox chemistry, deeper ocean waters are considered to have dominantly remained anoxic and ferruginous throughout this interval. Despite the persistence of ferruginous conditions, the mid-Proterozoic has long been considered notable for a general absence of iron formations (IFs), in stark contrast to the major intervals of IF deposition that occurred in the ferruginous oceans of the earlier Precambrian. In recent years, however, sporadic IFs have been found in the mid-Proterozoic, but controls on the genesis of these IFs, as well as implications for the evolution of ocean chemistry, remain poorly understood. Here, we present major and trace element data, iron speciation systematics, and organic carbon and iron isotope data for the ∼1.64 Ga Chuanlinggou Formation, which hosts a sudden reappearance of substantial IF deposition on the North China Craton. The Chuanlinggou IF has low Y/Ho ratios (24–26), slightly positive Eu anomalies (Eu/Eu* = 1.14–1.47), no Ce anomalies, slightly positive δ56Fe values (0.05 to 0.35 ‰), and high δ13Corg values (−29 ‰) relative to deeper water facies (down to −33.4 ‰). These observations provide support for a redox-stratified ocean with oxic surface waters overlying ferruginous deeper waters, and further suggest that IF deposition occurred in a stratified ocean with a shallow redoxcline during an interval of elevated Fe concentrations. Specifically, we suggest that the development of relatively intense ferruginous conditions was likely a consequence of hydrothermal activity during the breakup of the Columbia supercontinent. Microaerophilic iron-oxidizing bacteria and/or anoxygenic photosynthesizers likely played a key role in the generation of the Chuanlinggou IF. Importantly, however, data for shales that overly the Chuanlinggou IF suggest episodic intervals of deeper water oxygenation. Thus, similar to recent studies of the later Mesoproterozoic ocean, our data indicate that ocean chemistry during the latest Paleoproterozoic may have been more dynamic than previously considered, with fluctuating oxygen and iron levels likely exerting a limiting constraint on both the evolution of eukaryotes and the deposition of IFs across the immense interval of time encapsulated by the mid-Proterozoic.