We have been investigating the creation of novel proteins by means of block shuffling, where the term block refers to an amino acid sequence that corresponds to particular features of proteins, such as secondary structures, modules, functional motifs, and so on. Block shuffling makes it possible to explore the global sequence space, which is not feasible with conventional methods, such as DNA shuffling or family shuffling. To investigate what properties are required for the building blocks, we have analyzed the foldability and enzymatic activity of barnase mutants obtained by permutation of modules or secondary structure units. This reconstructive approach indicated that secondary structure units with mutual long-range interactions are more suitable than modules as building blocks, at least in the case of barnase. The results also suggested that proteins in evolutionarily intermediate states are created by block shuffling, and such proteins have the potential to be evolved into mature globular proteins. For the construction of combinatorial protein libraries, we have developed random multi-recombinant PCR (RM-PCR), which can combine different DNA fragments without homologous sequences. The libraries can be utilized for in vitro selection using in vitro virus (mRNA display) or stable (DNA display), which have also been developed in our laboratory. In this review article, we summarize our strategy to create novel proteins by block shuffling and review key literature in the field. Possible applications of the block shuffling strategy are also discussed.