The response of the mesolite crystal structure (Na16Ca16Al48Si72O240·64H2O) to dehydration was evaluated as a function of temperature and partial pressure of water (i.e., PH2O or relative humidity, RH) using laboratory X-ray powder diffraction (XRD; CuKα radiation) and synchrotron X-ray pair distribution function (PDF) methods. At 425 °C under low-PH2O conditions (PH2O ≤ ~1.3 mbar), dehydrated mesolite preserved the long-range ordered aluminosilicate framework structure, which has not been previously observed. This new dehydrated phase, x-metamesolite, has unit-cell parameters [a = 16.731(3) Å, b = 17.822(2) Å, c = 6.312(1) Å, V = 1882.5(5) Å3, and possible space group Fdd2] similar to those of other dehydrated natrolite phases (either α1- or α2-metanatrolite). Conversely, under high-PH2O conditions (PH2O > ~1.3 mbar), dehydrated mesolite became amorphous (amorphous T5O10) at 425 °C. The local structure of amorphous T5O10 was characterized by PDF analyses, which showed the formation of twisted T5O10 nano-fibers [with dimensions (LWH) of ~6.9 × 6.9 × 6.3 Å] resulting from breakage of the mesolite aluminosilicate framework. The two distinct high-temperature PH2O-dependent phase transition paths illustrate the importance of considering the combined effects of temperature and PH2O in mesolite. In addition, the low-temperature phase transition in mesolite, involving order-disorder of the extraframework cations, also showed a PH2O-dependent transition temperature. Although, there is no path dependence on PH2O for this transition, the local arrangement of Na, Ca, and vacancies in disordered metamesolite (formed through the extraframework cation order-disorder phase transition) likely influences the thermal stability of the aluminosilicate framework during further heating.