Charcot-Marie-Tooth disease (CMT) is a heterogenous group of hereditary peripheral nervous system disorder, with approximately twenty percent of the patients remaining genetically undiagnosed. Mutations in the sodium-potassium-ATPase alpha-1 polypeptide encoding ATP1A1 gene have been linked to CMT, however, the pathomechanisms underlying ATP1A1-related CMT have yet to be precisely elucidated. Despite we have identified a rare ATP1A1 c.620C>A; p.(Ser207Tyr) variant in a patient with late-onset axonal CMT, the functional implication of this S207Y protein remained unclear. This rare S207Y protein and three other known CMT-associated S207F, D811A, and G877S ATP1A1 protein were examined for their biochemical and biophysical properties. Utilizing bioinformatics analyses for the protein structure simulations, we observed that these CMT-relevant S207, S207, D811, and G877 loci occurred in the critical functional domains of the sodium-potassium pump. Biochemical investigation for these S207Y, S207F, D811A, and G877S proteins further validated a significant reduction of protein expression levels, protein instability with accelerated degradation, and aberrant membrane trafficking properties Electrophysiological evaluation by conducting two-electrode voltage clamp further verified the attenuated sodium-potassium co-transportation from these S207Y, S207F, D811A, and G877S sodium pumps. The ATP1A1 pump primarily expressed on the cell membrane, facilitating the establishment and maintenance of electrochemical gradients between intra- and extracellular environments, thereby influencing the excitability of neuronal and muscular cells. We also observed that the S207Y mutation engenders milder protein dysfunction, potentially correlating with the characteristic of later onset observed in our patients, thereby, comprehending the molecular characteristics of ATP1A1 mutations associated with hereditary peripheral nervous system disorders.