Copper nanoparticles (Cu-NPs) have a wide range of applications as heterogeneous catalysts. Here, we demonstrate a novel green biosynthesis route for producing Cu-NPs using the metal-reducing bacterium, Shewanella oneidensis. Thin section transmission electron microscopy (TEM) shows that the Cu-NPs are predominantly intracellular and present in a typical size range of 20 – 40 nm. Serial block-face scanning electron microscopy (SBFSEM) demonstrates the Cu-NPs are well-dispersed across the three dimensional (3D) structure of the cells. X-ray absorption near-edge spectroscopy (XANES) and extended x-ray absorption fine-structure spectroscopy (EXAFS) analysis show the nanoparticles are Cu(0), however, atomic resolution images and electron energy loss spectroscopy (EELS) suggest partial oxidation of the surface layer to Cu2O upon exposure to air. The catalytic activity of the Cu-NPs is demonstrated in an archetypal ‘click chemistry’ reaction, generating good yields during azide-alkyne cycloadditions (CuAAC), most likely catalyzed by the Cu(I) surface layer of the nanoparticles. Furthermore, cytochrome deletion mutants suggest a novel metal reduction system is involved in enzymatic Cu(II) reduction and Cu-NP synthesis, which is not dependent on the Mtr pathway commonly used to reduce other high oxidation state metals in this bacterium. This work demonstrates a simple, green synthesis method for producing efficient copper nanoparticle catalysts.