The germination process of seeds is influenced by the interplay between two opposing factors, pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI), which collectively regulate patterns of pectin methylesterification.  Despite the recognized importance of pectin methylesterification in seed germination, the specific mechanisms that govern this process remain unclear.  In this study, we demonstrated that the overexpression of GhPMEI53 is associated with a decrease in PME activity and an increase in pectin methylesterification.  This leads to seed cell wall softening, which positively regulates cotton seed germination.  AtPMEI19, the homologue in Arabidopsis thaliana, plays a similar role in seed germination to GhPMEI53, indicating a conserved function and mechanism of PMEI in seed germination regulation.  Further studies revealed that GhPMEI53 and AtPMEI19 directly contribute to promoting radicle protrusion and seed germination by inducing cell wall softening and reducing mechanical strength.  Additionally, the pathways of abscicic acid (ABA) and gibberellin (GA) in the transgenic materials showed significant changes, suggesting that GhPMEI53/AtPMEI19-mediated pectin methylesterification serves as a regulatory signal for the related phytohormones involved in seed germination.  In summary, GhPMEI53 and its homologs alter the mechanical properties of cell walls, which influence the mechanical resistance of the endosperm or testa.  Moreover, they impact cellular phytohormone pathways (e.g., ABA and GA) to regulate seed germination.  These findings enhance our understanding of pectin methylesterification in cellular morphological dynamics and signaling transduction, and contribute to a more comprehensive understanding of the PME/PMEI gene superfamily in plants.