Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H⁺-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H⁺-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H⁺-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H⁺-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.