Microtubules (MTs) are hollow biopolymeric filaments that function to define the shape of eukaryotic cells, serve as a platform for intracellular vesicular transport, and separate chromosomes during mitosis. One means of physiological regulation of MT mechanics and dynamics, critical to their adaptability in such processes, is through electrostatics due to the strong polyelectrolyte nature of MTs. Here, we show that in the presence of physiologically relevant amounts of divalent salts, MTs experience a dramatic increase in persistence length or stiffness, which is counter to theoretical expectations and experimental observations in similar systems (e.g., DNA). Divalent salt-dependent effects on MT dynamics were also observed with respect to suppressing depolymerization as well as reducing dispersion in kinesin-driven molecular motor transport assays. These results establish a novel mechanism by which MT dynamics, mechanics, and interaction with molecular motors may be regulated by physiologically relevant concentrations of divalent salts.