This study focuses on developing a framework for interpreting isotopic fractionations in the four stable sulfur isotopes, with a special focus on microbial effects. Calculations of low temperature equilibrium fractionations between various sulfur species were performed and suggest a tightly constrained fractionation relationship between the isotopes. This work was followed by conducting a series of batch culture experiments with two prominent sulfur metabolisms: sulfate reduction and sulfur disproportionation. The results of these experiments illustrate that measurable deviations from the equilibrium predictions exist, and further serve to highlight the ability of these measurements to differentiate between fractionations produced by these two metabolisms. This is a distinction not possible from traditional isotope measurements alone (³⁴S/³²S). In addition, models constructed to describe these observations provide insight into the intra-workings of these metabolic pathways, with the most recent experiments suggesting that the traditionally accepted model for isotope fractionation produced by sulfate reducers be re-evaluated. The microbial fractionations were also used to calibrate global sulfur cycle box models. These models have been coupled with the measurement of natural samples to illustrate the onset of microbial disproportionation ∼800 million years earlier in Earth history than previously thought and to describe the sulfur cycle of three Proterozoic marine basins.