The Neoproterozoic (1000-542 million years ago, Mya) was characterized by profound global environmental and evolutionary changes, not least of which included a major rise in atmospheric oxygen concentrations [1, 2], extreme climatic fluctuations and global-scale glaciation , and the emergence of metazoan life in the oceans [4, 5]. We present here phylogenomic (135 proteins and two ribosomal RNAs, SSU and LSU) and relaxed molecular clock (SSU, LSU, and rpoC1) analyses that identify this interval as a key transition in the marine nitrogen cycle. Specifically, we identify the Cryogenian (850-635 Mya) as heralding the first appearance of both marine planktonic unicellular nitrogen-fixing cyanobacteria and non-nitrogen-fixing picocyanobacteria (Synechococcus and Prochlorococcus ). Our findings are consistent with the existence of open-ocean environmental conditions earlier in the Proterozoic adverse to nitrogen-fixers and their evolution - specifically, insufficient availability of molybdenum and vanadium, elements essential to the production of high-yielding nitrogenases. As these elements became more abundant during the Cryogenian [7, 8], both nitrogen-fixing cyanobacteria and planktonic picocyanobacteria diversified. The subsequent emergence of a strong biological pump in the ocean implied by our evolutionary reconstruction may help in explaining increased oxygenation of the Earth's surface at this time, as well as tendency for glaciation. © 2014 Elsevier Ltd.