Hydrogen is a key energy carrier for a sustainable and low-carbon future, but its widespread deployment requires efficient purification and isotope separation technologies. Achieving high-purity hydrogen and separating isotopes such as deuterium and tritium remain challenging due to their similar physicochemical properties and low natural abundance. Metal–organic frameworks (MOFs), with their highly tunable pore structures and chemical functionalities, have emerged as advanced materials for selective hydrogen separation and purification. This chapter presents a comprehensive overview of MOF-based strategies for hydrogen and hydrogen isotope separation, emphasizing molecular sieving, kinetic quantum sieving, and chemical affinity quantum sieving mechanisms. Design principles, structure–property relationships, and representative experimental and theoretical studies are discussed to highlight the potential of advanced MOFs in addressing key challenges in hydrogen purification and isotope separation.