Photosynthetic purple non-sulfur bacteria (PNSB) can naturally convert electrons from organic compounds, protons from water, and energy from light into H2 gas, via the enzyme nitrogenase. In 2004, the first PNSB genome sequence was reported, that of Rhodopseudomonas palustris strain CGA009. The CGA009 genome sequence revealed natural attributes that favored H2 accumulation and revealed further potential for enhancing H2 production. Since then, the genomes of several more Rp. palustris species and other PNSB have been sequenced. Comparing these genomes has led to new ideas for improving the substrate range, rate, and photosynthetic efficiency of H2 production. Furthermore, systems biology or ‘omics’ approaches, including transcriptomics, proteomics, and fluxomics have been applied. Many of these systems level approaches have focused on the regulation and activity of nitrogenase – the enzyme responsible for H2 production. Guided by these approaches, metabolic engineering has targeted metabolic pathways that compete with H2 production for electrons, leading to strains with higher H2 yields and potentially linking the survival of these strains to the production of H2 biofuel. A systems level examination of PNSB is now turning to characterize largely unexplored but potentially crucial aspects involved in H2 production including non-coding small RNAs and post-translational modifications. Systems biology approaches are also being designed to eliminate experimenter bias and highlight genes of unknown function that contribute to H2 production, ideally providing clues to their function and their place in bacterial physiology. This chapter describes the contributions of systems biology to our understanding and application of H2 production by PNSB, focusing on Rhodopseudomonas palustris and referencing examples from other PNSB.