NO3- reduction is a metabolism that is widespread among ε-Proteobacteria and Aquificae, two abundant classes of microorganisms found at deep-sea vents. In this study, we used Sulfurovum lithotrophicum, Caminibacter mediatlanticus and Thermovibrio ammonificans as representatives of these groups to study ecophysiological, metabolic and biogeochemical parameters associated with chemolithoautotrophic NO3- reduction under different temperature regimes. We observed that while S. lithotrophicum and C. mediatlanticus achieved higher cell densities than T. ammonificans, the overall NO3- consumption by the latter was on average ∼ 9 and ∼ 5 times faster on a per cell basis, respectively. Comparison with previously published data from other cultured vent ε-Proteobacteria and Aquificae suggests that the rate-yield trade-offs observed in our experiments are generally conserved between these two groups in line with their ecophysiologies. Kinetic isotope effects of N from NO3- reduction were 9.6 ± 2.7 ‰ for S. lithotrophicum, 6.4 ± 0.7 ‰ for C. mediatlanticus and 8.8 ± 0.6 ‰ for T. ammonificans. Our results help evaluate how metabolic partitioning between growth efficiency and reaction kinetics during chemolithoautotrophic NO3- reduction affect the concentration and isotope composition of N compounds at deep-sea hydrothermal vents.