Aqueous zinc-iodine batteries (AZIBs) are promising candidates for energy storage systems due to the low cost and inherent safety of the zinc anode, iodine cathode, and aqueous electrolytes. However, the uneven Zn 2+ flux through the separator induces non-uniform Zn deposition at the anode, leading to dendritic growth. This dendritic growth eventually results in short-circuit, limiting the performance and stability of AZIBs. Furthermore, polyiodide dissolution at the cathode can lead to the migration of polyiodide species through the separator to the anode, causing self-discharge or shuttle effects. These challenges arise from unregulated ion transport in the electrolyte, highlighting the importance of the separator in controlling ion transport to enhance cell performance and cyclability. However, conventional glass fiber (GF) separators with nonuniform macropores cannot effectively regulate Zn 2+ flux and suppress polyiodide migration, necessitating the development of a dual-functional separator. To resolve these issues, metal–organic framework (MOF) interlayers are introduced on both sides of the GF separator using a dip coating method. The uniform distribution of nano-porous MOF particles is verified by scanning electron microscopy. The MOF has an internal pore size of 0.48 nm, effectively regulating the Zn 2+ flux and suppressing the migration of polyiodide (0.6–1.1 nm). The computational simulation confirms that the well-ordered nanopores of the interlayer successfully redistribute and homogenize Zn 2+ flux at the electrolyte–anode interface. Furthermore, the nanoporous MOF layer improves the Zn²⁺ transference number, resulting in a Zn 2+ conductivity of 2.2 mS cm −1 , which is sufficiently high for practical application in AZIBs. Chronoamperometry and linear sweep voltammetry measurements reveal that the MOF layer facilitates efficient 3D Zn nucleation and suppresses hydrogen evolution, thereby stabilizing the Zn anode. Blocking of polyiodide crossover through the MOF interlayer is further effectively achieved, as revealed by UV-vis spectroscopy. A Zn metal || C@I 2 cell with MOF interlayer exhibits higher capacity retention after rest compared to cells with the GF separator, indicating that the MOF layer effectively suppresses self-discharge by preventing polyiodide crossover to the Zn anode. A Zn-symmetric cell with MOF interlayer shows stable cycling performance with uniform Zn morphologies and a small amount of residual by-products. In addition, the Zn metal || C@I 2 cell with MOF interlayer exhibits superior charge–discharge operation with a discharge capacity of 142.5 mAh g −1 at 2C, demonstrating the efficacy of the MOF interlayer. The interlayer design proposed in this study would enable the practical use of high-safety and high-energy-density AZIBs.