Rhodobacter sphaeroides produces hydrogen gas (H2) from organic compounds via nitrogenase under anaerobic-light conditions in the presence of poor nitrogen sources, such as l-glutamate. R. sphaeroides utilizes the ethylmalonyl-coenzyme A (EMC) pathway for acetate assimilation, but its H2 yield from acetate in the presence of l-glutamate has been reported to be low. In this study, the deletion of ccr encoding crotonyl-coenzyme A (crotonyl-CoA) carboxylase/reductase, a key enzyme for the EMC pathway in R. sphaeroides, revealed that the EMC pathway is essential for H2 production from acetate and l-glutamate but not for growth and acetate consumption in the presence of l-glutamate. We introduced a plasmid expressing aceBA from Rhodobacter capsulatus encoding two key enzymes for the glyoxylate bypass into R. sphaeroides, which resulted in a 64% increase in H2 production. However, compared with the wild-type strain expressing heterologous aceBA genes, the strain with aceBA introduced in the genetic background of an EMC pathway-disrupted mutant showed a lower H2 yield. These results indicate that a combination of the endogenous EMC pathway and a heterologously expressed glyoxylate bypass is beneficial for H2 production. In addition, introduction of the glyoxylate bypass into a polyhydroxybutyrate (PHB) biosynthesis-disrupted mutant resulted in a delay in growth along with H2 production, although its H2 yield was comparable to that of the wild-type strain expressing heterologous aceBA genes. These results suggest that PHB production is important for fitness to the culture during H2 production from acetate and l-glutamate when both acetate-assimilating pathways are present.IMPORTANCE As an alternative to fossil fuel, H2 is a promising renewable energy source. Although photofermentative H2 production from acetate is key to developing an efficient process of biohydrogen production from biomass-derived sugars, H2 yields from acetate and l-glutamate by R. sphaeroides have been reported to be low. In this study, we observed that in addition to the endogenous EMC pathway, heterologous expression of the glyoxylate bypass in R. sphaeroides markedly increased H2 yields from acetate and l-glutamate. Therefore, this study provides a novel strategy for improving H2 yields from acetate in the presence of l-glutamate and contributes to a clear understanding of acetate metabolism in R. sphaeroides during photofermentative H2 production.