Alternative sigma (σ) factors, proteins that recruit RNA polymerase core enzyme to target promoters, are one mechanism by which bacteria transcriptionally regulate groups of genes in response to environmental stimuli. A class of σ(70) proteins, termed extracytoplasmic function (ECF) σ factors, are involved in cellular processes such as bacterial stress responses and virulence. Here, we describe an ECF16 σ factor, EcfS (Blr4928) from the gram-negative soil bacterium Bradyrhizobium japonicum USDA110, that plays a critical role in the establishment of a functional symbiosis with soybean. Nonpolar insertional mutants of ecfS form immature nodules that do not fix nitrogen, a defect that can be successfully complemented by expression of ecfS. Overexpression of the cocistronic gene, tmrS (blr4929), phenocopies the ecfS mutant in planta and, therefore, we propose that TmrS is a negative regulator of EcfS, a determination consistent with the prediction that it encodes an anti-σ factor. Microarray analysis of the ecfS mutant and tmrS overexpressor was used to identify 40 transcripts misregulated in both strains. These transcripts primarily encode proteins of unknown and transport-related functions and may provide insights into the symbiotic defect in these strains.