a b s t r a c tThe Wonoka-Shuram Anomaly represents the largest negative carbon isotope excursion recognized in the geologic record and is associated with the emergence and diversification of metazoan life ca. 580 million years ago (Ma). The origin of the anomaly is highly debated, with interpretations ranging from primary to diagenetic, each having unique and potentially transformative implications for early life. Here, we apply carbonate clumped isotope thermometry to three sections expressing the anomaly in order to constrain mineral formation temperatures and thus directly calculate water oxygen isotope compositions (␦ 18 O w ) with which carbonate minerals equilibrated. With ␦ 18 O w known, it is possible to address previous hypotheses for the origin of the anomaly. In each section, precipitation temperatures correlate positively with reconstructed ␦ 18 O w . Previous hypotheses, based on the covariance of ␦ 18 O carb vs. ␦ 13 C carb (uncorrected for temperature effects), suggested a meteoric diagenetic origin for the anomaly. However, reconstructed ␦ 18 O w values do not covary with carbon isotope compositions (␦ 13 C carb ) within anomaly facies. Rather, the oxygen isotope and temperature data are consistent with carbonate recrystallization and equilibration under increasingly rock-buffered conditions. Based on simple modeling and comparison to modern formation fluids, recrystallization may have occurred in an environment far removed from the initial depositional or early diagenetic regime. In addition, although clumped isotope temperatures vary significantly and reach elevated values consistent with burial diagenesis, it is unclear to what degree, if at all, carbon isotope values were reset during recrystallization. Ultimately, these new data indicate that Wonoka-Shuram-aged carbonates experienced equilibration with fluids under increasingly closedsystem conditions. The clumped isotope data do not provide a means to distinguish previous hypotheses outright, but provide additional context for the evaluation of geochemical signatures within these ancient carbonate rocks.