Voltage‐gated proton channels (HV1) have been found in many mammalian cells and play a crucial role in the immune system, male fertility, and cancer progression. Glycosaminoglycans play a significant role in various aspects of cell physiology, including the modulation of membrane receptors and ion channel function. We present here evidence that mechanosensitivity of the dimeric HV1 channel transduce changes on cell membrane fluidity related to the defective biosynthesis of chondroitin sulfate and heparan sulfate in Chinese Hamster Ovary (CHO‐745) cells into a leftward shift in the activation voltage dependence. This effect was accompanied by an increase in the H+ current, and an acceleration of the activation kinetics, under symmetrical or asymmetrical pH gradient (ΔpH) conditions. Similar gating alterations were evoked by two naturally occurring HV1 N‐terminal truncated isoforms expressed in wild‐type CHO‐K1 and CHO‐745 cells. On three different monomeric HV1 constructs, no alterations in the biophysical parameters were observed. Moreover, we have shown that HV1 gating can be modulated by manipulating CHO‐K1 cell membrane fluidity. Our results suggest that the defective biosynthesis of chondroitin sulfate and heparan sulfate on CHO‐745 cell increases membrane fluidity and allosterically modulates the coupling between voltage‐ and ΔpH‐sensing through the dimeric HV1 channel.