Cohesin exhibits DNA loop extrusion activity when bound to an ATPase activator Scc2 (NIPBL in humans), thereby organizing higher-order chromosome folding. In budding yeast, the majority of the chromosome-bound cohesins lack association with Scc2. It remains unknown how the interaction between Scc2 and cohesin is regulated on the chromosome and what physiological consequences malfunction in this regulatory mechanism causes. Here, we show that simultaneous deletion of Wpl1 and Eco1, two of the known cohesin regulators, resulted in Scc2 co-localization with cohesin around the centromeres of metaphase chromosomes. In these cells, the pericentromeric DNA loops were enlarged to connect the centromere to the cohesin/Scc2 co-bound sites at distances of up to a few hundred kb, indicating highly active loop extrusion by the Scc2-associated cohesin. Furthermore, we demonstrated that deltawpl1 deltaeco1 cells exhibited a delay in the progression of mitotic chromosome segregation, a phenotype dependent on the presence of Scc2 in metaphase. These findings suggest that Wpl1 and Eco1 cooperatively regulate Scc2-cohesin interaction on chromosomes, restrict the size of the pericentromeric DNA loops, and facilitate mitotic chromosome segregation.