Transcriptional enhancers in the cell nuclei typically interact with the target promoters in cis over long stretches of chromatin, but the mechanism of this communication remains unknown. Previously we have developed a defined in vitro system for quantitative analysis of the rate of distant enhancer-promoter communication (EPC) and have shown that the chromatin fibers maintain efficient distant EPC in cis. Here we investigate the roles of linker histone H1 and HMGN5 protein in EPC. A considerable negative effect of histone H1 on EPC depending on its C- and N-tails was shown. Protein HMGN5 that affects chromatin compaction and is associated with active chromatin counteracts EPC inhibition by H1. The data suggest that the efficiency of the interaction between the enhancer and the promoter depends on the structure and dynamics of the chromatin fiber localized between them and can be regulated by proteins associated with chromatin.