ABSTRACT Chromosome spatial organization and dynamics influence DNA-related metabolic processes. SMC complexes like cohesin are essential instruments of chromosome folding. Cohesin-dependent chromatin loops bring together distal loci to regulate gene transcription, DNA repair and V(D)J recombination processes. Here we characterize further the roles of members of the cohesin holocomplex in regulating chromatin loop expansion, showing that Scc2, which stimulates cohesin ATPase activity, is essential for the translocation process required to extend DNA loop length. Eco1-dependent acetylation of Smc3 during S phase counteracts this activity through the stabilization of Pds5, to finely tune loop sizes and stability during G2. Inhibiting Pds5 in G2 leads to a strong enlargement of pre-established, stable DNA loops, in a Scc2-dependent manner. Altogether, the study strongly supports a Scc2-mediated translocation process driving expansion of DNA loops in living cells.