Branch migration (BM) of DNA Holliday junctions (HJs) occurs through base-pair rearrangements between homologous DNA molecules during the repair of double-strand breaks (DSBs) by homologous recombination. Despite the fact that BM is conserved among organisms and is essential to stabilize recombination intermediates, which occurs by avoiding the reversal of strand exchange leading to the faithful repair of DSBs, molecular insights into the BM process including kinetics, the effects of microenvironments, and the role of HJ-binding proteins are poorly understood. In this article, using single-molecule fluorescence analysis of a synthetic, mobile HJ as a model system, we systematically investigated the effects of cell-mimic solvent composition and crowding on BM by varying the concentration of cosolutes, namely dimethyl sulfoxide (DMSO) and poly(ethylene glycol) (PEG). The single-molecule analyses revealed that BM is affected by cosolutes in a concentration-dependent manner. In addition, the kinetic analysis of the fluorescence resonance energy transfer (FRET) traces showed that the BM is significantly accelerated under the crowding environments. The finding that the mobility of the HJ is enhanced by cell-like environments not only provides a better insight into BM but also may open up new avenues for targeting HJs and the associated BM process for possible therapeutic interventions.