The kernel water concentration (KWC) varies more intensely during the physiological dehydration phase, and the dehydration process during this phase has been demonstrated to be closely associated with internal physiological processes in maize kernels. To better illustrate the physiological dehydration process (PDP) of maize kernels, moisture characteristic parameters were consecutively recorded after pollination, and the timing of the occurrence of net water loss was confirmed. Additionally, a tandem mass tag (TMT) based proteomic analysis was performed to identify the significantly differentially expressed proteins during the critical period of the PDP (PDP cp). The results showed that maize varieties presented significant variance in the KWC, kernel water content, dry-down rate (DR) and absolute DR; nevertheless, the KWC was similar (below 65%) when net water loss occurred. In addition, 20-28 days after pollination was indicated to be the PDP cp in this study. Based on proteome analysis, Yuyu 30 and Suyu 41 showed 163 and 132 differentially expressed proteins (DEPs), respectively during the PDP cp and 99 DEPs overlapped between the varieties. During the PDP cp , the desiccation tolerance, antioxidation capability, detoxification and other stress response abilities of maize kernels were significantly increased. The increased defense abilities enabled the maize kernels to establish coordinated, diverse, stable defense mechanisms that accelerated water loss from maize kernels during the PDP cp .