Coordinated trafficking of intracellular vesicles is of critical importance for the maintenance of cellular health and homeostasis. Members of the Rab GTPase family serve as master regulators of vesicular trafficking, maturation, and fusion by reversibly associating with distinct target membranes and recruiting specific effector proteins. Rabs act as molecular switches by cycling between an active, GTP-bound form and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Rab7 specifically regulates the trafficking and maturation of vesicle populations that are involved in protein degradation including late endosomes, lysosomes, and autophagic vacuoles. Missense mutations of Rab7 cause a dominantly-inherited axonal degeneration known as Charcot-Marie-Tooth type 2B (CMT2B) through an unknown mechanism. Patients with CMT2B present with length-dependent degeneration of peripheral sensory and motor neurons that leads to weakness and profound sensory loss. To gain insight into the pathogenesis of CMT2B, we undertook extensive characterization of two disease-causing Rab7 mutants, L129F and V162M. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. We further demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect as previously suggested, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Using an unbiased proteomics approach, we characterize effector interactions in wild-type and mutant Rab7 and identify several novel Rab7 interactors. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that increased activation of mutant Rab7 is counterbalanced by unregulated, GTP-hydrolysis-independent membrane cycling. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease by misregulation of native Rab7 activity. Future experiments will address the impact of Rab7 misregulation on neuronal trafficking and trophic signaling.