We investigated the influence of organic substrates and phosphate concentration on the rates of dissimilatory microbial sulfate reduction and the 34S/32S isotopic fractionation produced by several Desulfovibrio species. Our experiments corroborate the previously reported species-specific correlation between sulfur isotope fractionation and cell-specific sulfate reduction rates. We also identify cell size as a key factor that contributes to the species-effect of this correlation. Phosphate limitation results in larger cells and contributes to a small decrease in sulfur isotope fractionation concomitant with an apparent increase in cell-specific sulfate reduction rates. Sulfur isotope fractionation in phosphate-limited cultures asymptotically approaches a lower limit of approximately 5‰ as cell-specific sulfate reduction rates increase to >100 fmol cell−1 day−1. These experimental results test models that link the reversibilities of enzymatic steps in dissimilatory sulfate reduction to sulfur isotope fractionation and show that these models can provide consistent predictions across large variations in physiological states experienced by sulfate reducing bacteria.