The utility of the intramolecular Nozaki-type coupling for the synthesis of macrocyclic benzo[b]thiophene-fused enediynes has been explored. The starting acyclic enediynes were prepared by the iodocyclization of 2-(buta-1,3-diynyl)thioanisoles followed by the Sonogashira cross-coupling of the resulting iodo-substituted benzo[b]thiophene with corresponding acetylenes. We found that Cr(II)-promoted intramolecular cyclization of 7-[2-(iodoethynyl)benzo[b]thiophen-3-yl]hept-6-ynal and 7-[3-(iodoethynyl)benzo-[b]thiophen-2-yl]hept-6-ynal resulted in the formation of 11-membered macrocyclic enediynes, while both expected 10-membered enediynes cannot be produced under the Nozaki-type reaction from corresponding 6-[3-(iodoethynyl)benzo[b]thiophen-2-yl]hex-5-ynal and 6-[2-(iodoethynyl)benzo[b]thiophen-3-yl]hex-5-ynal. In the case the reaction was catalyzed by Ni(II), the attack on a proximal triple bond led to the formation of 2-methylenecycloalkane-1-ol fragments, instead of macrocyclization. The DFT analysis of the ring strain in the benzo[b]thiophene-fused 10- and 11-membered enediyne-containing cycle provides the plausible explanation of the observed regioselectivity.