<p>The Cell Wall Integrity (CWI) MAPK pathway in <i>Saccharomyces cerevisiae</i> is crucial for coordinating adaptive responses to cell wall stress. We investigated the effects of zymolyase (ZY), a cell wall-perturbing agent, on cell morphology and cell separation. Our findings reveal that sublethal ZY treatment transiently delays mother-daughter cell separation, leading to multibudded cell clusters. This phenotype, which is accompanied by initiation of a new budding cycle before cell separation is complete, was observed with cell wall perturbations but not with osmotic, oxidative, or ER stress. Genetic and molecular analyses indicate that this cell separation defect depends on phosphorylation-dependent activation of Slt2, the MAP kinase of the CWI pathway. The phenotype is not fully mediated by the primary transcriptional regulator of the pathway, Rlm1, suggesting that Slt2-dependent outputs beyond canonical Rlm1 transcription contribute to the response. We also show that ZY treatment is associated with altered spatiotemporal localization and dynamics of septin rings, as well as changes in the intracellular trafficking of key septal proteins. Notably, delivery of the endochitinase Cts1 to the bud neck is delayed, and the residence time of the exocyst subunit Exo84 and the myosin Myo2 at the septum is reduced. Despite altered protein trafficking, the transcriptional program governed by the daughter-specific transcription factor Ace2 remains largely unaffected. Our data are consistent with a cell-separation delay resulting from a coordinated response involving the CWI pathway, which correlates with changes in septin dynamics and cytokinetic protein trafficking. These findings, which suggest a mechanism that transiently halts cell separation when cell wall integrity is compromised, highlight a link between cell wall homeostasis and the regulation of cell division in yeast.</p>