Most gaseous oxygen (O2) on Earth is produced via oxygenic photosynthesis. However, new evidence indicates that O2 is also produced in permanently dark ecosystems. This so-called “dark oxygen production” (DOP) can proceed via abiotic chemical reactions or via microbial dismutation of chlorite and nitric oxide—metabolisms fundamentally different from photosynthesis. Recent work suggests that microbial DOP is widespread in groundwater ecosystems, yet definitive evidence is lacking to date. Our team has the abilities, instruments, and expertise to provide such evidence and elucidate the origins, processes, and production rates of groundwater O2. We hypothesize that microbial DOP is a globally relevant process in groundwater ecosystems today, and that groundwater aquifers represent model systems to study O2 production and consumption in the deep geologic past, as well as in subsurface ecosystems of other celestial bodies. We propose an international, multi-disciplinary project to provide comprehensive insights into the geochemistry, microbiology, and ecology of DOP. Our combined expertise leverages three key technological advances: (1) triple- and clumped-oxygen isotope analyses, which can unambiguously link O2 to biological DOP origins (led by PI Hemingway), (2) determination of DOP rates and fluxes at unprecedented resolution using nanomolar O2 concentration measurements (led by PI Kraft), and (3) high-throughput analyses of O2-producing enzymes and pathways present in complex communities using genome sequencing and protein mass spectrometry (led by PI Ruff). We will apply these techniques to a sample set from diverse aquifers spanning a broad range of ecosystems in Canada, Finland, Germany, Switzerland, and the USA. We expect several key outcomes. First, we will determine the first ever triple- and clumped-oxygen isotope signatures of DOP by different biotic and abiotic sources, values that are needed to decipher natural signals. Second, we will use stable isotope-labelling to determine rates of microbial DOP in groundwaters, critical measurements that are lacking to date. Finally, we will conduct the first global survey of key microbial lineages and genes to understand the diversity, abundance, and activity of microbes involved in DOP. These insights are crucial to understand groundwater aquifers, which represent Earth’s largest source of drinking water.