Heterologous expression of nitrogenase has been actively pursued because of the far-reaching impact of this enzyme on agriculture, energy and environment. Yet, isolation of an active two-component, metallocentre-containing nitrogenase from a non-diazotrophic host has yet to be accomplished. Here, we report the heterologous synthesis of an active Mo-nitrogenase by combining genes from Azotobacter vinelandii and Methanosarcina acetivorans in Escherichia coli. Metal, activity and EPR analyses demonstrate the integrity of the metallocentres in the purified nitrogenase enzyme; whereas growth, nanoSIMS and NMR experiments illustrate diazotrophic growth and 15N enrichment by the E. coli expression strain, as well as accumulation of extracellular ammonia upon deletion of the ammonia transporter that permits incorporation of thus-generated N into the cellular mass of a non-diazotrophic E. coli strain. As such, this study provides a crucial prototype system that could be optimized/modified to enable future transgenic expression and biotechnological adaptations of nitrogenase.