Biological nitrogen fixation constitutes the main input of fixed nitrogen to Earth s ecosystems, and its isotope effect is a key parameter in isotope-based interpretations of the N cycle. The nitrogen isotopic composition (δ15N) of newly fixed N is currently believed to be∼-10/00, based on measurements of organic matter from diazotrophs using molybdenum (Mo)-nitrogenases. We show that the vanadium (V)- and iron (Fe)-only "alternative" nitrogenases produce fixed N with significantly lower δ15N (-6 to -70/00). An important contribution of alternative nitrogenases to N2 fixation provides a simple explanation for the anomalously low δ15N (<-20/00) in sediments from the Cretaceous Oceanic Anoxic Events and the Archean Eon. A significant role for the alternative nitrogenases over Mo-nitrogenase is also consistent with evidence of Mo scarcity during these geologic periods, suggesting an additional dimension to the coupling between the global cycles of trace elements and nitrogen.