IMPORTANCE Photoheterotrophic bacteria, like Rhodopseudomonas palustris, obtain energy from light and carbon from organic compounds during anaerobic growth. Cells can naturally produce the biofuel H2 as a way of disposing of excess electrons. Unexpectedly, feeding cells organic compounds with more electrons does not necessarily result in more H2. Despite repeated observations over the last 40 years, the reasons for this discrepancy have remained unclear. In this paper, we identified two metabolic factors that influence the H2 yield, (i) the route taken to make biosynthetic precursors and (ii) the amount of CO2-fixing Calvin cycle flux that competes against H2 production for electrons. We show that the H2 yield can be improved on all substrates by using a strain that is incapable of Calvin cycle flux. We also contributed quantitative knowledge to the long-standing question of why photoheterotrophs must produce H2 or fix CO2 even on relatively oxidized substrates.