We measured the δ98Mo of cells and media from molybdenum (Mo) assimilation experiments with the freshwater cyanobacterium Anabaena variabilis, grown with nitrate as a nitrogen (N) source or fixing atmospheric N2. This organism uses a Mo-based nitrate reductase during nitrate utilization and a Mo-based dinitrogenase during N2 fixation under culture conditions here. We also demonstrate that it has a high-affinity Mo uptake system (ModABC) similar to other cyanobacteria, including marine N2-fixing strains. Anabaena variabilis preferentially assimilated light isotopes of Mo in all experiments, resulting in fractionations of −0.2‰ to −1.0‰ ± 0.2‰ between cells and media (εcells–media), extending the range of biological Mo fractionations previously reported. The fractionations were internally consistent within experiments, but varied with the N source utilized and for different growth phases sampled. During growth on nitrate, A. variabilis consistently produced fractionations of −0.3 ± 0.1‰ (mean ± standard deviation between experiments). When fixing N2, A. variabilis produced fractionations of −0.9 ± 0.1‰ during exponential growth, and −0.5 ± 0.1‰ during stationary phase. This pattern is inconsistent with a simple kinetic isotope effect associated with Mo transport, because Mo is likely transported through the ModABC uptake system under all conditions studied. We present a reaction network model for Mo isotope fractionation that demonstrates how Mo transport and storage, coordination changes during enzymatic incorporation, and the distribution of Mo inside the cell could all contribute to the total biological fractionations. Additionally, we discuss the potential importance of biologically incorporated Mo to organic matter-bound Mo in marine sediments.