In prior work, we observed that soybean (Glycine max L. cv Merr.) seeds inoculated with a mutant Bradyrhizobium japonicum strain unable to catabolize Pro (Pro dehydrogenase− [ProDH−]) resulted in plants that, when forced to depend on N2 fixation as the sole source of nitrogen and subjected to mild drought stress, suffered twice as large a loss in seed yield as did plants inoculated with the parental strain. Here, we used a continuous gas flow system to measure H2 evolution as a function of time and leaf water potential (ΨL). Since one H2 is produced for every N2 fixed as an obligate part of the mechanism of N2 fixation, these measurements serve as the basis for continuous monitoring of the N2 fixation rate. In five replicate experiments, the slope of the decline in N2 fixation rate in response to water stress was always greater for plants inoculated with the mutant strain unable to catabolize Pro or take up H2 (ProDH−, hup−) than it was for plants inoculated with the parental strain (ProDH+, hup−). In aggregate, the probability that this difference occurred by chance alone was 0.005. In combination with the earlier result, this is consistent with bacteroid catabolism of Pro synthesized in response to mild drought stress having a positive impact on N2 fixation rate and seed yield.