Separation of racemic mixtures is of great importance and interest in chemistry and pharmacology. Porous materials including metal-organic frameworks (MOFs) have been widely explored as chiral stationary phases (CSPs) in chiral resolution. However, it remains a challenge to develop new CSPs for reversed-phase high-performance liquid chromatography (RP-HPLC), which is the most popular chromatographic mode and accounts for over 90% of all separations. Here we demonstrated for the first time that highly stable Zr-based MOFs can be efficient CSPs for RP-HPLC. By elaborately designing and synthesizing three tetracarboxylate ligands of enantiopure 1,1'-biphenyl-20-crown-6, we prepared three chiral porous Zr(IV)-MOFs with the framework formula [Zr6O4(OH)8(H2O)4(L)2]. They share the same flu topological structure but channels of different sizes and display excellent tolerance to water, acid, and base. Chiral crown ether moieties are periodically aligned within the framework channels, allowing for stereoselective recognition of guest molecules via supramolecular interactions. Under acidic aqueous eluent conditions, the Zr-MOF-packed HPLC columns provide high resolution, selectivity, and durability for the separation of a variety of model racemates, including unprotected and protected amino acids and N-containing drugs, which are comparable to or even superior to several commercial chiral columns for HPLC separation. DFT calculations suggest that the Zr-MOF provides a confined microenvironment for chiral crown ethers that dictates the separation selectivity.