Tau is an intrinsically disordered neuronal protein known to modulate microtubule dynamics through the suppression of dynamic instability. Recent studies have shown that Tau mediates dynamical linear microtubule bundles similar to microtubule fascicles observed in vivo, which are a cardinal feature of the axon initial segment (P. J. Chung, C. Song, et al. Nature Communications 2016, 7, 12278. DOI: 10.1038/ncomms12278 and ACS Macro Lett. 2018, 7, 228-232. DOI: 10.1021/acsmacrolett.7b00937). In order to better understand tau's role in the physiologic bundling of MTs, quantitative techniques of synchrotron small-angle x-ray scattering data—combined with plastic-embedded TEM, and other analytical techniques—have been utilized. Here we show that Ca2+ and Mg2+ in the mM concentration range (corresponding to the average concentration in cells for Mg2+) can destabilize steady-state, bundled microtubules (in the presence of GTP at 37 °C), inducing a novel bundle-to-bundle transition prior to the tau-coated tubulin ring state. This transitory bundled phase suggests that tubulin oligomers also play a role in stabilizing MT bundles.