As an indispensable part of electric devices, separator play a key role in electrochemical performance. Thus, separator engineering is a promising way of constructing better energy storage and conversion systems. MXenes, a novel class of 2D transition metal nitrides, carbides, and carbonitrides, are emerging for separator engineering due to their unique properties, such as high conductivity, large surface area, controllable hydrophilicity, abundant surface chemistries, and superior mechanical performance. The related investigations have remarkably increased since 2020. However, comprehensive reviews on this topic are rare. Herein, an overview of MXenes in separator engineering for rechargeable batteries is systematically presented. Firstly, the fundamental principles of MXenes, including composition, synthesis techniques, and functionalization, are summarized. Subsequently, the basic information on separator design and its optimization is introduced. Finally, the applications of MXenes for separator engineering and their applications in rechargeable batteries are reviewed. In the end, the perspectives and challenges on the further development of MXenes-based separators in rechargeable batteries are proposed. This review will help researchers in the design and construction of functional separators for batteries, supercapacitors, fuel cells, etc.