Connexin 32 (Cx32) is a fundamental protein in the peripheral nervous system as its mutations are the leading cause of the demyelinating X-linked form of Charcot–Marie–Tooth neuropathy (CMT1X). Recent findings suggest that ATP release by transient opening of unpaired Cx32 channels (hemichannels) in Schwann cells participates in nerve myelination, but it is unknown if CMT1X mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening in physiological conditions. Using a combination of electrophysiology and fluorescence optical microscopy, we found that lack of the C-terminal domain in Cx32 carrying a known CMT1X mutation (R220X) inhibited hemichannel opening during stimulation by a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Complete restoration of the Ca2+-dependent gating was obtained in R220X hemichannels by both extracellular and intracellular application of a peptide that mimics the Cx32 cytoplasmic loop. Molecular dynamics simulations suggest that both the C-terminus in the wild-type hemichannel and the peptide in the R220X one may have thermodynamic stability effects on the cytoplasmic loop that would otherwise abnormally fluctuate in their absence. In accordance with previous reports, R220X hemichannels also displayed a reduced sensitivity to transmembrane voltage that, in view of our single channel recordings, we attributed to a reduced number of subconductance states. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels displayed similar values of the unitary conductance of the fully open state as well as of the unitary permeabilities to cAMP and Lucifer yellow. Taken together, our data support the hypothesis that paracrine signalling alteration by mutant Cx32 hemichannels underlies CMT1X pathogenesis and point to possible therapeutic interventions for the neuropathy caused by the R220X mutation.