Separation of alkane isomers represents a crucial process in the petrochemical industry in order to achieve high octane rating of gasoline. Herein, we report the first example of complete separation of linear, monobranched and dibranched alkane isomers by a single adsorbent. A calcium-based robust microporous metal-organic framework, Ca(H2tcpb) (tcpb = 1,2,4,5-tetrakis(4-carboxyphenyl)-benzene) exhibits unique molecular exclusion behavior which enables full separation of binary or ternary mixtures of alkane isomers into pure form of each isomerate. The successful separation of monobranched and dibranched hexane isomers will not only lead to the production of higher quality gasoline with maximum possible octane numbers but also fill the gap in the current separation technology. Exploration of separation mechanism indicates that structural flexibility and adsorbate-dependent structure change of the porous framework plays a vital role for the observed temperature-dependent molecular sieving property of the adsorbent.