NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides complex I enzymes (complex I-A and complex I-E) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex I-A) or NADH oxidation (complex I-E). The canonical alphaproteobacterial complex I isozyme (complex I-A) was also shown to be important for routing electrons to nitrogenase-mediated H-2 production, while the horizontally acquired enzyme (complex I-E) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains.IMPORTANCECells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.