We hereby report a strategy to synthesize sequence-regulated substituted polyacetylenes using living anionic polymerization of designed monomers, that is, 2,4-disubstituted butadienes. It is found that proper substituents, such as 2-isopropyl-4-phenyl, lead to nearly 100% 1,4-addition during the polymerization, thus, giving product with high regioregularity, precise molecular weight, and narrow molecular weight distribution. The product is convertible into sequence-regulated substituted polyacetylene by oxidative dehydrogenation using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Block copolymers containing polyacetylene segment are also prepared. Owing to the versatility of the anionic reactions, the present strategy can serve as a powerful tool of precise control on polymer chain microstructure, architecture, and functionalities in the same time.